Methods, Compounds, and Compositions For Treatment and Prophylaxis in the Respiratory Tract

ABSTRACT

The present invention provides a method of reducing the quantity of mucus in the respiratory tract of a subject with elevated levels of mucus in said respiratory tract. The method includes administering to the subject a compound or composition containing a therapeutically effective amount of a fusion protein comprising a sialidase or an active portion thereof and an anchoring domain. The therapeutically effective amount comprises an amount of the fusion protein that results in a reduction of the quantity of mucus in the respiratory tract after administration of the compound or composition when compared to the quantity of mucus present prior to administration of the compound or composition.

CLAIM OF PRIORITY

This application claims the benefit of U.S. patent application Ser. No.15/136,751, filed on Apr. 22, 2016, which claims the benefit of U.S.patent application Ser. No. 14/489,428, filed on Sep. 17, 2014, whichclaims the benefit of U.S. patent application Ser. No. 13/769,095, filedon Feb. 15, 2013, which claims the benefit of U.S. patent applicationSer. No. 12/940,742, filed on Nov. 5, 2010, (U.S. Pat. No. 8,398,971)which claims the benefit of U.S. Provisional Patent Application Ser. No.61/259,033, filed on Nov. 6, 2009, and U.S. Provisional PatentApplication Ser. No. 61/259,055, filed on Nov. 6, 2009, U.S. ProvisionalPatent Application Ser. No. 61/322,813, filed on Apr. 9, 2010, U.S.Provisional Patent Application Ser. No. 61/332,063, filed on May 6,2010, and U.S. Provisional Patent Application Ser. No. 61/381,420 filedon Sep. 9, 2010, the entire contents of each of which are herebyincorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contract numberHHSN266200600015C awarded by the United States Department of Health andHuman Services, National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND

Respiratory tract infections (RTIs) are among the most common, andpotentially most severe, types of infectious diseases. Examples of RTIsinclude influenza, parainfluenza, RSV, sinusitis, otitis, laryngitis,bronchitis and pneumonia.

One common feature of agents that cause RTIs, such as respiratorypathogenic bacteria, is that they establish commensal colonization onthe mucosal surface of the upper airway; such colonization precedes aninfection and generally is prerequisite for infections. Bacterialcolonization in a neonate occurs shortly after birth. During one'slifetime, the upper airway, specifically the nasopharynx and oropharynx,remains a dynamic ecological reservoir of microbial species withbacteria being acquired, eliminated and re-acquired continually. In mostcases, the bacterial flora in the pharynx are harmless. However, whenthe condition of the host is altered, some microorganisms may invadeadjacent tissues or bloodstream to cause diseases.

In addition to serving as the port of entry for mucosal and invasiveinfections by both bacteria and viruses, the nasopharynx and oropharynxare also the major source of spreading the pathogenic microorganismsbetween individuals, as well as the reservoir where antibiotic-resistantbacteria are selected (Garcia-Rodriguez and Martinez, J AntimicrobChemother, (2002) 50(Suppl S2), 59-73; Soriano and Rodriguez-Cerrato, JAntimicrob Chemother, (2002) 50 Suppl S2, 51-58). It is well establishedclinically that individuals who are prone to RTIs tend to be persistentand recurrent carriers of pathogenic bacteria (Garcia-Rodriguez andMartinez, J Antimicrob Chemother, (2002) 50(Suppl S2), 59-73; Mbaki etal., Tohoku J Exp. Med., (1987) 153(2), 111-121). For example,Helicobacter pylori is a human pathogen implicated in gastritis andpeptic ulcer. The bacterium resides in the human stomach and binds toepithelial cells of the gastric antrum.

Other disorders of the respiratory tract (more broadly termed, RTDs) maynot be caused by infectious agents, although they could arise as aconsequence of infection. Examples of RTDs include a variety ofobstructive lung diseases such as allergic and non-allergic asthma,COPD, bronchiectasis, vasculitis, mucous plugging, Wegener'sgranulomatosis and cystic fibrosis (CF). RTDs can have a genetic basis(for example, CF), can arise due to immunodeficiencies, can arise due toother deficiencies (for example, alpha-1-antitrypsin deficiency can makepeople more susceptible to bronchiectasis), can be caused by allergensand/or chemical pollutants, or can present as complications of otherinfectious diseases such as the RTIs described above or inflammatorydiseases such as inflammatory bowel syndrome or Crohn's disease.

Common indications of RTIs and RTDs include inflammation and elevatedlevels of mucous in the respiratory tract. However, currently availabledrugs that are used to treat RTIs and RTDs often are unable toameliorate these associated conditions. For example, Relenza® is awell-known treatment for influenza, but it is not recommended forpatients who suffer from underlying airway disease, such as asthma andCOPD. Thus, in addition to the need for drugs that reduce inflammationand/or reduce mucus in the respiratory tract or limit its increase aredrugs that are capable of treating respiratory infectious diseases, suchas influenza, parainfluenza and RSV, without aggravating underlyingrespiratory conditions, such as asthma, bronchitis, bronchiectasis, andCOPD, of patients.

The present invention recognizes that drugs currently available formedical use have limited efficacy with respect to reducing inflammation,and/or reducing mucus in the respiratory tract or limiting its increasein the respiratory tract, and those that are available are associatedwith side effects. The present invention also recognizes that there is aneed for drugs for treating respiratory infectious diseases in patientswith underlying airway disease, such as asthma, bronchitis,bronchiectasis and COPD. Thus, there is a need for new drugs that areable to reduce inflammation, and/or drugs that reduce mucus in therespiratory tract or limit its increase in the respiratory tract. Thereis also a need for drugs that can treat respiratory infectious diseaseswhile reducing inflammation, and/or while reducing mucus in therespiratory tract or limiting its increase in the respiratory tract.

SUMMARY

The compositions, components of compositions and methods provided beloware characterized by a variety of component ingredients, steps ofpreparation, and biophysical, physical, biochemical or chemicalparameters. As would be apparent to those of skill in the art, thecompositions and methods provided herein include any and allpermutations and combinations of the ingredients, steps and/orparameters described below.

The invention relates to the use of therapeutic compounds andcompositions that have anti-inflammatory effects in the respiratorytract and to methods of treating respiratory inflammation andprophylaxis against respiratory inflammation. The invention also relatesto therapeutic compounds and compositions that can be used to prevent ortreat diseases that are caused by, cause, or are exacerbated byrespiratory inflammation, including, but not limited to, inflammationnot caused by allergies or allergic reactions.

The invention also relates to the use of therapeutic compounds andcompositions to reduce the quantity of mucus in the respiratory tract ofsubjects with elevated levels of mucus in their respiratory tracts, andto corresponding methods of treatment. The invention also relates to theuse of therapeutic compounds and compositions to limit an increase inthe quantity of mucus in the respiratory tract of subjects above abaseline level of mucus in their respiratory tract and to correspondingmethods of treatment. The invention also relates to therapeuticcompounds and compositions that can be used to prevent or treatconditions and/or diseases that are caused by, cause, or are exacerbatedby increased mucus in the respiratory tract, such as, both allergic andnon-allergic asthma, chronic obstructive pulmonary disease (COPD),bronchitis (both acute and non-acute), bronchiectasis, cystic fibrosis(CF), vasculitis, mucus plugging, Wegener's granulomatosis, pneumonia,tuberculosis, cancers involving the lungs or the respiratory tract,Kartagener syndrome, Young's syndrome, chronic sinopulmonary infections,alpha 1-antitrypsin deficiency, primary immunodeficiencies, acquiredimmune deficiency syndrome, opportunistic infections, infectious andpost infectious states, common cold, exercise-induced asthma, exerciseinduced hypersecretion of mucus, inflammatory bowel disease, ulcerativecolitis, Crohn's disease, allergic reactions to inhaled fungus spores,respiratory infections, respiratory obstructions, inhalation oraspiration of ammonia and other toxic gases, pulmonary aspiration,alcoholism, various allergies, and any other disorder that causesincreased mucus production in the respiratory tract or is caused by orexacerbated by increased mucus production in the respiratory tract. Insome embodiments, the subject has more than one of the aforementionedconditions and/or diseases. In other embodiments, the subject having oneor more of the aforementioned conditions and/or diseases does not havean accompanying infectious disease (RTI), such as influenza,parainfluenza or RSV. In other embodiments, the subject having one ormore of the aforementioned conditions and/or diseases has one or moreaccompanying infectious diseases, such as influenza, parainfluenza orRSV. Thus, provided herein are methods, compounds and compositions fortreating inflammatory and/or allergic responses associated with an RTI,an RTD, or combinations thereof.

The compounds and compositions provided herein can reduce mucusproduction in the respiratory tract and/or reduce the levels ofinflammatory cells that cause allergic or non-allergic types ofinflammation, including, without limitation, monocytes, macrophages,dendritic cells, histiocytes, Kuppfer cells, mastocytes andneutrophiles.

The compounds and compositions provided herein include a sialidase oractive portion thereof. Without being bound by any theory, sialic acidshave been implicated in allergic and/or inflammatory responsesassociated with RTIs and RTDs. For example, siglecs (sialic acid bindingIg-like lectins) are members of the immunoglobulin (Ig) superfamily thatbind to sialic acid and are mainly expressed by cells of thehematopoietic system. At least 11 siglecs have been discovered and theyseem to exclusively recognize cell surface sialic acid as the ligand. Itis believed that the binding of siglecs to sialic acid mediatescell-cell adhesion and interactions (Crocker and Varki, Trends Immunol.,(2001) 22(6), 337-342; Angata and Brinkman-Van der Linden, Biochim.Biophys. Acta, (2002) 1572(2-3), 294-316). Siglec-8 (SAF-2) is anadhesion molecule that is highly restricted to the surface ofeosinophils, basophils, and mast cells, which are the central effectorcells in allergic conditions including allergic rhinitis, asthma andeczema. Siglec-8 (homologous to Siglec-F in mice) is considered to beresponsible for mediating the recruitment of the three allergic celltypes to the airway, the lungs and other sites of allergy. Siglec-1(sialoadhesion) and siglec-2 (CD22) are the adhesion molecules onmacrophages and B cells, both types of cells play central roles inimmune reactions that lead to inflammation. Siglec-9 is predominantlyexpressed on neutrophils, which are known to be important effector cellsin inflammation (von Gunten, Yousefi, Seitz, Jakob, Schaffner, Seger,Takala, Villiger, and Simon (2005) Blood 106:1423-1431). Further,without being bound by any particular theory, sialic acid residues havebeen implicated in the interaction of muscaranic receptors withagonists; thus, sialidases can affect the interecation of muscarinicreceptors with their agonists.

The present invention provides a method of reducing the quantity ofmucus in the respiratory tract of a subject with elevated levels ofmucus in said respiratory tract. The method includes administering tothe subject a compound or composition containing a therapeuticallyeffective amount of a fusion protein having a sialidase or an activeportion thereof and an anchoring domain. The therapeutically effectiveamount includes an amount of the fusion protein that results in areduction of the quantity of mucus in the respiratory tract afteradministration of the compound or composition when compared to thequantity of mucus present prior to administration of the composition.

In another embodiment, another method of reducing the quantity of mucusin the respiratory tract of a subject with elevated levels of mucus insaid respiratory tract is provided. The method includes administering tothe subject a compound or composition containing a therapeuticallyeffective amount of a fusion protein. The fusion protein has at leastone catalytic domain of a sialidase, wherein the catalytic domain of thesialidase includes the sequence of amino acids extending from amino acid274 to amino acid 666 of SEQ ID NO:12, inclusive, and at least oneanchoring domain. The anchoring domain can be a glycosaminoglycan (GAG)binding domain of human amphiregulin including the amino acid sequenceof SEQ ID NO:7. The therapeutically effective amount includes an amountof the fusion protein that results in a reduction of the quantity ofmucus in the respiratory tract after administration of the compound orcomposition when compared to the quantity of mucus present prior toadministration of the composition.

In another embodiment, another method of reducing the quantity of mucusin the respiratory tract of a subject with elevated levels of mucus insaid respiratory tract is provided. The method includes administering tothe subject a compound or composition containing a therapeuticallyeffective amount of a protein or peptide having a sialidase or an activeportion thereof. The therapeutically effective amount includes an amountof the protein or peptide that results in a reduction of the quantity ofmucus in the respiratory tract after administration of the compound orcomposition when compared to the quantity of mucus present prior toadministration of the compound or composition.

In another embodiment, a method of treating or ameliorating the effectsof chronic obstructive pulmonary disease (COPD), bronchitis,bronchiectasis, cystic fibrosis (CF), vasculitis, mucus plugging,Wegener's granulomatosis, pneumonia, tuberculosis, cancer involving thelungs or the respiratory tract, Kartagener syndrome, Young's syndrome,chronic sinopulmonary infection, alpha 1-antitrypsin deficiency, primaryimmunodeficiency, acquired immune deficiency syndrome, opportunisticinfection, an infectious state, a post infectious state, common cold,exercise induced hypersecretion of mucus, inflammatory bowel disease,ulcerative colitis, Crohn's disease, respiratory infection, respiratoryobstruction, inhalation or aspiration of a toxic gas, pulmonaryaspiration, or alcoholism in a subject with an elevated level of mucusin his or her respiratory tract is provided. The method includesadministering to the subject a compound or composition containing atherapeutically effective amount of a fusion protein. The fusion proteinhas at least one catalytic domain of a sialidase, wherein the catalyticdomain of the sialidase includes the sequence of amino acids extendingfrom amino acid 274 to amino acid 666 of SEQ ID NO:12, inclusive, and atleast one anchoring domain. The anchoring domain can be aglycosaminoglycan (GAG) binding domain of human amphiregulin includingthe amino acid sequence of SEQ ID NO:7. The therapeutically effectiveamount includes an amount of the fusion protein that results in areduction of the quantity of mucus in the respiratory tract afteradministration of the compound or composition when compared to thequantity of mucus present prior to administration of the compound orcomposition.

In another embodiment, another method of treating or ameliorating theeffects of chronic obstructive pulmonary disease (COPD), bronchitis,bronchiectasis, cystic fibrosis (CF), vasculitis, mucus plugging,Wegener's granulomatosis, pneumonia, tuberculosis, cancer involving thelungs or the respiratory tract, Kartagener syndrome, Young's syndrome,chronic sinopulmonary infection, alpha 1-antitrypsin deficiency, primaryimmunodeficiency, acquired immune deficiency syndrome, opportunisticinfection, an infectious state, a post infectious state, common cold,exercise induced hypersecretion of mucus, inflammatory bowel disease,ulcerative colitis, Crohn's disease, respiratory infection, respiratoryobstruction, inhalation or aspiration of a toxic gas, pulmonaryaspiration, or alcoholism in a subject with an elevated level of mucusin his or her respiratory tract is provided. The method includesadministering to the subject a compound or composition containing atherapeutically effective amount of a fusion protein. The fusion proteinhas a sialidase or an active portion thereof and an anchoring domain.The therapeutically effective amount includes an amount of the fusionprotein that results in a reduction of the quantity of mucus in therespiratory tract after administration of the compound or compositionwhen compared to the quantity of mucus present prior to administrationof the compound or composition.

In another embodiment, another method of treating or ameliorating theeffects of chronic obstructive pulmonary disease (COPD), bronchitis,bronchiectasis, cystic fibrosis (CF), vasculitis, mucus plugging,Wegener's granulomatosis, pneumonia, tuberculosis, cancer involving thelungs or the respiratory tract, Kartagener syndrome, Young's syndrome,chronic sinopulmonary infection, alpha 1-antitrypsin deficiency, primaryimmunodeficiency, acquired immune deficiency syndrome, opportunisticinfection, an infectious state, a post infectious state, common cold,exercise induced hypersecretion of mucus, inflammatory bowel disease,ulcerative colitis, Crohn's disease, respiratory infection, respiratoryobstruction, inhalation or aspiration of a toxic gas, pulmonaryaspiration, or alcoholism in a subject with an elevated level of mucusin his or her respiratory tract is provided. The method includesadministering to the subject a compound or composition containing atherapeutically effective amount of a protein or peptide having asialidase or an active portion thereof. The therapeutically effectiveamount includes an amount of the protein or peptide that results in areduction of the quantity of mucus in the respiratory tract afteradministration of the compound or composition when compared to thequantity of mucus present prior to administration of the compound orcomposition.

In another embodiment, a method of limiting an increase in the quantityof mucus in the respiratory tract of a subject above a baseline level ofmucus in said subject's respiratory tract is provided. The methodincludes administering to the subject a compound or compositioncontaining a therapeutically effective amount of a fusion protein. Thefusion protein has at least one catalytic domain of a sialidase, whereinthe catalytic domain of the sialidase includes the sequence of aminoacids extending from amino acid 274 to amino acid 666 of SEQ ID NO:12,inclusive, and at least one anchoring domain, wherein the anchoringdomain is a glycosaminoglycan (GAG) binding domain of human amphiregulincomprising the amino acid sequence of SEQ ID NO:7. The therapeuticallyeffective amount includes an amount of the fusion protein that limits anincrease in the quantity of mucus in the respiratory tract of saidsubject above a baseline level after administration of the compound orcomposition.

In another embodiment, another method of limiting an increase in thequantity of mucus in the respiratory tract of a subject above a baselinelevel of mucus in said subject's respiratory tract is provided. Themethod includes administering to the subject a compound or compositioncontaining a therapeutically effective amount of a fusion protein havinga sialidase or an active portion thereof and an anchoring domain. Thetherapeutically effective amount includes an amount of the fusionprotein that limits an increase in the quantity of mucus in therespiratory tract of said subject above a baseline level afteradministration of the compound or composition.

In yet another embodiment, another method of limiting an increase in thequantity of mucus in the respiratory tract of a subject above a baselinelevel of mucus in said subject's respiratory tract is provided. Themethod includes administering to the subject a compound or compositioncontaining a therapeutically effective amount of a protein or peptidehaving a sialidase or an active portion thereof. The therapeuticallyeffective amount includes an amount of the protein or peptide thatlimits an increase in the quantity of mucus in the respiratory tract ofthe subject above a baseline level after administration of the compoundor composition.

Also contemplated herein are methods of identifying sialidases or activeportions thereof according to the compounds or compositions providedherein, where the sialidases or active portions thereof are effective atreducing the quantity of mucus in the respiratory tract of subjects. Thereduction in mucus can be measured directly in standard assays known tothose of skill in the art. For example, in some embodiments, a singlecompound or a library or collection of compounds or compositionscomprising sialidase(s) and/or catalytically active portion(s) thereofare administered to an animal model of asthma having an associatedinflammatory response, such as the guinea pig and the mouse as describedin Example 1 and Example 2, respectively. An asthmatic or otherinflammatory condition is created in the animal whereby the accumulationof mucus in the lung or respiratory tract is increased. The level ofmucus is then quantitated and compared to the level after treatment witha sialidase or active portion thereof If there is a reduction of themucus level in the presence of the sialidase or active portion thereof,the sialidase or active portion thereof is identified or selected as onethat can be used in the methods provided herein for treatinginflammation, allergies and/or associated inflammatory/allergicresponses, such as the overproduction of mucus.

In some embodiments, a sialidase or active portion thereof according tothe compounds and compositions provided herein is identified as beingsuitable for treating inflammation, allergies or associated responses bymeasuring its ability to disrupt muscarinic receptor-agonistinteractions according to standard methods known to those of skill inthe art. For example, provided herein is a method of assessing whether acompound or composition comprising a sialidase and/or catalyticallyactive portion thereof reduces the quantity of mucus in the respiratorytract of a subject, by

(a) contacting the muscarinic receptors of an animal subject with acompound or composition that includes a sialidase and/or a catalyticallyactive portion thereof;

(b) administering a muscarinic receptor agonist to the subject;

(c) quantitating the airway resistance in the subject;

(d) comparing the airway resistance level measured in (c) with theairway resistance in the absence of contact with the compound orcomposition;

(e) identifying whether the compound or composition reduces the airwayresistance relative to the airway resistance in the absence of contactwith the compound or composition; and

(f) if the compound or composition reduces the airway resistance asdetermined in (e), assessing the compound or composition as one thatreduces the quantity of mucus in the respiratory tract of the subject.Such a method is exemplified in Example 3.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows GAG-binding sequences of four human genes: PF4, humanplatelet factor 4; IL8, human interleukin 8; AT III, human antithrombinIII; ApoE, human apolipoprotein E; AAMP, human angio-associatedmigratory cell protein; human amphiregulin.

FIG. 2 is a sequence comparison between human sialidases NEU2 and NEU4(SEQ ID NOs: 8 & 9).

FIG. 3 is a table comparing substrate specificity of bacterial andfungal sialidases.

FIG. 4 depicts the nucleotide and amino acid sequence (SEQ ID NOs: 28 &29) of a construct of the present invention encoding His6-AvCD. NcoI andHindIII sites used for cloning into pTrc99a are shown in bold.

FIG. 5 depicts the nucleotide and amino acid sequences (SEQ ID NOs: 18 &19) of another construct of the present invention encoding AR-AvCD. NcoIand HindIII sites used for cloning into pTrc99a are shown in bold.

FIG. 6 depicts the nucleotide and amino acid sequences (SEQ ID NO: 36 &37) of another construct of the present invention encoding AR-G₄S-AvCD.NcoI and HindIII sites used for cloning into pTrc99a are shown in bold.

FIGS. 7A-B are graphs showing that topical administration of recombinantAR-AvCD sialidase fusion protein reduces the inflammatory responses offerrets infected with an influenza A (H1N1) virus. FIG. 7A shows thetotal number of inflammatory cells from nasal wash samples obtained frominfected animals at the indicated times after infection. The proteinconcentration was determined in cell-free nasal wash samples of infectedferrets. Infected ferrets were vehicle-treated (squares) or were treatedwith recombinant AR-AvCD sialidase fusion protein made from Construct #2(triangles). Uninfected animals were also treated with recombinantAR-AvCD sialidase fusion protein (diamonds). Statistically significantvalues are labeled with * (p<0.05) and ** (p<0.01).

FIG. 8 provides graphs showing formula and explanation of the EnhancedPause (PENH), the parameter used for measuring bronchoconstriction inconscious unrestrained animals.

FIG. 9 provides a graph showing early asthmatic reaction in response toan OVA-aerosol. Results are expressed as arithmetic average±SEM.*p<0.05, ***p<0.001 using student's t-test.

FIG. 10 provides a graph showing the total number of cells in guineapigs on the day of section. Results are expressed as arithmeticaverage±SEM. **p<0.01, ***p<0.001 using student's t-test.

FIG. 11 provides a graph showing the total number of macrophagesrecovered in guinea pig BAL fluid on the day of section. Results areexpressed as arithmetic average±SEM. **p<0.01.

FIG. 12 provides a graph showing the total number of lymphocytesrecovered in guinea pig BAL fluid on the day of section. Results areexpressed as arithmetic average 35 SEM. *p<0.05.

FIG. 13 provides a graph showing the total number of neutrophilsrecovered in guinea pig BAL fluid on the day of section. Results areexpressed as arithmetic average±SEM. *p<0.05, ***p<0.001.

FIG. 14 provides a graph showing the total number of eosinophilsrecovered in guinea pig BAL fluid on the day of section. Results areexpressed as arithmetic average±SEM. *p<0.05, ***p<0.001.

FIG. 15 provides a graph showing the percent change in Penh at Mch 48mg/mL in the effect of sialidase treatment on the early and lateasthmatic reaction in guinea pigs.

FIG. 16 provides a graph showing the percent change in Pehn at a rangeof Mch concentrations in the effect of sialidase treatment on the earlyand late asthmatic reaction in guinea pigs.

FIG. 17 provides a graph showing blood Eosinophils in the effect ofsialidase treatment on the early and late asthmatic reaction in guineapigs.

FIG. 18 provides a graph showing PAS staining for lung mucus in theeffect of sialidase treatment on the early and late asthmatic reactionin guinea pigs.

FIGS. 19A-F provide a PAS staining for lung mucus.

FIG. 20 provides a graph showing MBP immunostaining for eosinophils inthe effect of sialidase treatment on the early and late asthmaticreaction in guinea pigs.

FIGS. 21A-B provide graphs showing reduced airway resistance in naivemice treated intranasally with low doses of DAS181 (methacholinechallenged).

FIG. 22 provides a graph showing reduced airway resistance in naive micetreated intranasally with a low dose of DAS181 (methacholinechallenged).

FIG. 23 provides a graph showing reduced airway resistance in naive micetreated intranasally with DAS181 (carbachol challenged).

FIG. 24 provides a graph showing airway resistance in naive mice treatedintranasally with a low dose of DAS185 (methacholine challenged).

FIG. 25 provides graphs showing time-course of DAS185 mediated reductionof airway resistance (methacholine challenged).

FIG. 26 provides a graph showing reduced airway resistance in naive micetreated intranasally with very lose doses of DAS181 (methacholinechallenged).

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The present disclosure provides, inter alia, novel methods of use forcompounds described in U.S. patent application Ser. Nos. 10/718,986 and10/939,262 (both of which are hereby incorporated by reference in theirentirety) to reduce mucus, e.g., in the respiratory tract of subjectswith elevated levels of mucus in their respiratory tract. In someembodiments, the present disclosure provides compositions and methodsfor reducing mucus (e.g., mucus levels) in a subject in need of reducedmucus levels and that does not have influenza (e.g., is not infectedwith influenza at the time of treatment) or asthma.

In some embodiments, the compounds can include compounds made by NexBio,Inc. under the compound name DAS181 and under the trademark Fludase®(provided herein as SEQ ID NO:21). DAS181 is a fusion protein comprisinga catalytic domain of a sialidase, and an anchoring domain. Several ofthe examples described herein use DAS 181 or compositions containing DAS181.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the manufacture or laboratory procedures described beloware well known and commonly employed in the art. Conventional methodsare used for these procedures, such as those provided in the art andvarious general references. Where a term is provided in the singular,the inventors also contemplate the plural of that term. Where there arediscrepancies in terms and definitions used in references that areincorporated by reference, the terms used in this application shall havethe definitions given herein. As employed throughout the disclosure, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

As used herein, a “subject” includes any animal for whom diagnosis,screening, monitoring or treatment is contemplated. Animals includemammals such as primates and domesticated animals. An exemplary primateis human. A patient refers to a subject such as a mammal, primate, humanor livestock subject afflicted with a disease condition or for which adisease condition is to be determined or risk of a disease condition isto be determined.

In some embodiments, the methods disclosed herein can include selectinga subject in need of reduced mucus levels and that is not infected withone or more of influenza, parainfluenza, and/or respiratory syncytialvirus (RSV). In some instances, the terms infected or infection caninclude the presence of a influenza and/or parainfluenza virus and/orRSV in a subject. In some instances, the terms infected or infection caninclude the presence of active or replicating influenza and/orparainfluenza virus and/or RSV in a subject. In some embodiments, asubject with an active or replicating influenza and/or parainfluenzavirus and/or RSV infection can be selected based on the presence ordetection of influenza and/or parainfluenza virus shedding and/or RSVshedding in the subject (e.g., in a sample from the subject). In someembodiments, the methods disclosed herein can include selecting asubject in need of reduced mucus levels, wherein the subject has alatent influenza, parainfluenza, and/or RSV infection.

An “animal model” as used herein means an animal that sufficientlymimics, resembles or reproduces a disease or condition of interest inits anatomy, physiology, or response (to a pathogen or allergen, e.g.)so as to be useful in medical research that can be extrapolated to thedisease or condition of interest (e.g., to screen for diagnostic ortherapeutic agents; to measure therapeutic efficacy of a compound orcomposition, etc.). For example, the guinea pig and the mouse can beanimal models to mimic inflammatory and/or allergic responses associatedwith asthma, as demonstrated in Examples 1 and 2, respectively. Themouse also can be an animal model to study the interaction of muscarinicreceptors with their agonists, and the disruption thereof by agents suchas the compounds and compositions provided herein (see Example 3).

A “pathogen” can be any virus or microorganism that can infect a cell, atissue or an organism. A pathogen can be a virus, bacterium, orprotozoan.

A “target cell” is any cell that can be infected by a pathogen or anycell that can interact with inflammatory cells, or a host cell that isthe intended destination for an exogenous gene transferred by arecombinant virus.

“Inflammatory cells” are the cells that carry out or participate ininflammatory responses of the immune system. Inflammatory cells includeB lymphocytes, T lymphocytes, macrophages, basophils, eosinophils, mastcells, NK cells, monocytes, and neutrophils.

An “extracellular activity that can inhibit adhesion or function ofinflammatory cells” is any activity that can prevent inflammatory cellsfrom contacting the target cell and affecting the normal physiologicalstatus of the target cell.

A “domain that can anchor said at least one therapeutic domain to themembrane of a target cell”, also called an “extracellular anchoringdomain” or simply, “anchoring domain” refers to a chemical entity canthat can stably bind a moiety that is at or on the exterior of a cellsurface or is in close proximity to the surface of a cell. Anextracellular anchoring domain can be reversibly or irreversibly linkedto one or more moieties, such as one or more therapeutic domains, andthereby cause the one or more attached therapeutic moieties to beretained at or in close proximity to the exterior surface of aeukaryotic cell. An extracellular anchoring domain can bind at least onemolecule on the surface of a target cell or at least one molecule foundin close association with the surface of a target cell. For example, anextracellular anchoring domain can bind a molecule covalently ornoncovalently associated with the cell membrane of a target cell, or canbind a molecule present in the extracellular matrix surrounding a targetcell. An extracellular anchoring domain can be a peptide, polypeptide,or protein, and can also comprise any additional type of chemicalentity, including one or more additional proteins, polypeptides, orpeptides, a nucleic acid, peptide nucleic acid, nucleic acid analogue,nucleotide, nucleotide analogue, small organic molecule, polymer,lipids, steroid, fatty acid, carbohydrate, or a combination of any ofthese.

As used herein, a protein or peptide sequences is “substantiallyhomologous” to a reference sequence when it is either identical to areference sequence, or comprises one or more amino acid deletions, oneor more additional amino acids, or more one or more conservative aminoacid substitutions, and retains the same or essentially the sameactivity as the reference sequence. Conservative substitutions may bedefined as exchanges within one of the following five groups:

I. Small, aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,Pro, Gly

II. Polar, negatively charged residues and their amides: Asp, Asn, Glu,Gln

III. Polar, positively charged residues: His, Arg, Lys

IV. Large, aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys

V. Large aromatic residues: Phe, Try, Trp

Within the foregoing groups, the following substitutions are consideredto be “highly conservative”: Asp/Glu, His/Arg/Lys, Phe/Tyr/Trp, andMet/Leu/Ile/Val. Semi-conservative substitutions are defined to beexchanges between two of groups (I)-(IV) above which are limited tosupergroup (A), comprising (I), (II), and (III) above, or to supergroup(B), comprising (IV) and (V) above. In addition, where hydrophobic aminoacids are specified in the application, they refer to the amino acidsAla, Gly, Pro, Met, Leu, Ile, Val, Cys, Phe, and Trp, whereashydrophilic amino acids refer to Ser, Thr, Asp, Asn, Glu, Gln, His, Arg,Lys, and Tyr.

A “sialidase” is an enzyme that can remove a sialic acid residue from asubstrate molecule. The sialidases (N-acylneuraminosylglycohydrolases,EC 3.2.1.18) are a group of enzymes that hydrolytically remove sialicacid residues from sialo-glycoconjugates. Sialic acids are alpha-ketoacids with 9-carbon backbones that are usually found at the outermostpositions of the oligosaccharide chains that are attached toglycoproteins and glycolipids. One of the major types of sialic acids isN-acetylneuraminic acid (Neu5Ac), which is the biosynthetic precursorfor most of the other types. The substrate molecule can be, asnonlimiting examples, an oligosaccharide, a polysaccharide, aglycoprotein, a ganglioside, or a synthetic molecule. For example, asialidase can cleave bonds having alpha(2,3)-Gal, alpha(2,6)-Gal, oralpha(2,8)-Gal linkages between a sialic acid residue and the remainderof a substrate molecule. A sialidase can also cleave any or all of thelinkages between the sialic acid residue and the remainder of thesubstrate molecule. Two major linkages between Neu5Ac and thepenultimate galactose residues of carbohydrate side chains are found innature, Neu5Ac alpha (2,3)-Gal and Neu5Ac alpha (2,6)-Gal. Both Neu5Acalpha (2,3)-Gal and Neu5Ac alpha (2,6)-Gal molecules can be recognizedby influenza viruses as the receptor, although human viruses seem toprefer Neu5Ac alpha (2,6)-Gal, avian and equine viruses predominantlyrecognize Neu5Ac alpha (2,3)-Gal. A sialidase can be anaturally-occurring sialidase, an engineered sialidase (such as, but notlimited to a sialidase whose amino acid sequence is based on thesequence of a naturally-occurring sialidase, including a sequence thatis substantially homologous to the sequence of a naturally-occurringsialidase). As used herein, “sialidase” can also mean the active portionof a naturally-occurring sialidase, or a peptide or protein thatcomprises sequences based on the active portion of a naturally-occurringsialidase.

A “fusion protein” is a protein comprising amino acid sequences from atleast two different sources. A fusion protein can comprise amino acidsequence that is derived from a naturally occurring protein or issubstantially homologous to all or a portion of a naturally occurringprotein, and in addition can comprise from one to a very large number ofamino acids that are derived from or substantially homologous to all ora portion of a different naturally occurring protein. In thealternative, a fusion protein can comprise amino acid sequence that isderived from a naturally occurring protein or is substantiallyhomologous to all or a portion of a naturally occurring protein, and inaddition can comprise from one to a very large number of amino acidsthat are synthetic sequences.

A “sialidase catalytic domain protein” is a protein that comprises thecatalytic domain of a sialidase, or an amino acid sequence that issubstantially homologous to the catalytic domain of a sialidase, butdoes not comprises the entire amino acid sequence of the sialidase thecatalytic domain is derived from, wherein the sialidase catalytic domainprotein retains substantially the same activity as the intact sialidasethe catalytic domain is derived from. A sialidase catalytic domainprotein can comprise amino acid sequences that are not derived from asialidase, but this is not required. A sialidase catalytic domainprotein can comprise amino acid sequences that are derived from orsubstantially homologous to amino acid sequences of one or more otherknown proteins, or can comprise one or more amino acids that are notderived from or substantially homologous to amino acid sequences ofother known proteins.

“Therapeutically effective amount” means an amount of a composition orcompound that is needed for a desired therapeutic, prophylactic, orother biological effect or response when a composition or compound isadministered to a subject in a single dosage form. The particular amountof the composition or compound will vary widely according to conditionssuch as the nature of the composition or compound, the nature of thecondition being treated, the age and size of the subject.

“Treatment” means any manner in which one or more of the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of thecomposition or compound herein, such as for reducing mucus in therespiratory tract.

“Respiratory tract” means the air passages from the nose to thepulmonary alveoli, including the nose, throat, pharynx, larynx, trachea,and bronchi, and it also includes the lungs, and is sometimes referredto by medical practitioners as the respiratory system.

“Inhaler” means a device for giving medicines in the form of a spray ordry powder that is inhaled (breathed in either naturally or mechanicallyforced in to the lungs) through the nose or mouth, and includes withoutlimitation, a passive or active ventilator (mechanical with or with anendotracheal tube), nebulizer, dry powder inhaler, metered dose inhaler,and pressurized metered dose inhaler.

“Inhalant” is any substance that is inhaled through the nose or mouth.

“Reducing the quantity of mucus” means diminishing all or some,generally more than by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more of the amountof mucus in the respiratory tract when compared with the amount prior toadministration of the compositions or compounds described herein.“Reducing the quantity of mucus” can also mean reducing the amount ofmucus in an amount that is observable by a healthcare practitioner usingwhatever medical implements are available for such observation, such as,e.g., by auscultation, by MM or other radiographic study, by directvisualization with a bronchoscope or other visualization device, or bymeasuring patient mucus over time. “Reducing the quantity of mucus” canalso mean reducing the amount of mucus in an amount that is observableby the patient or subject himself or herself with self-reporting orself-observation, such as, e.g., monitoring the amount of expectoratedor swallowed mucus over time, or by subjectively observing the sense ofcongestion in his or her lungs over time.

“Limiting an increase in the quantity of mucus” means that the amount ofmucus in the respiratory tract after administration of the compositionsand compounds described herein does not increase more than if they hadnot been administered. “Limiting an increase in the quantity of mucus”also means that the amount of mucus in the respiratory tract afteradministration of the compositions and compounds described herein doesnot increase after their administration of the compositions andcompounds. “Limiting an increase in the quantity of mucus” can also meanlimiting an increase over the patient's baseline at the time ofadministration of the compounds or compositions in an amount that isobservable or ascertainable by a healthcare practitioner using whatevermedical implements and analytical systems are available for suchobservations, such as, e.g., by ausculation, by MM or other radiographicstudy, by direct visualization with a bronchoscope or othervisualization device, or by measuring patient sputum over time.“Limiting an increase in the quantity of mucus” can also mean limitingan increase over the patient's baseline at the time of administration ofthe compounds or compositions in an amount that is observable by thepatient or subject himself or herself with self-reporting orself-observation, such as, e.g., monitoring the amount of expectoratedor swallowed mucus over time, or by subjectively observing the sense ofcongestion in his or her lungs over time.

“Excipient” as used herein means one or more inactive substances orcompounds that either alone or in combination are used as a carrier forthe active ingredients of a medication. As used herein “excipient” canalso mean one or more substances or compounds that are included in apharmaceutical composition to improve its beneficial effects or thathave a synergistic effect with the active ingredient.

Peptide or Protein Based Compounds

The present invention includes peptide or protein-based compounds thatcomprise at least one domain that can anchor the compound to themembrane of a eukaryotic cell and at least one additional domain that isa therapeutic domain. By “peptide or protein-based” compounds, it ismeant that the two major domains of the compound have an amino acidframework, in which the amino acids are joined by peptide bonds. Apeptide or protein-based compound can also have other chemical compoundsor groups attached to the amino acid framework or backbone, includingmoieties that contribute to the anchoring activity of the anchoringdomain, or moieties that contribute to the therapeutic activity of thetherapeutic domain. For example, the protein-based therapeutics used inthe present invention can comprise compounds and molecules such as butnot limited to: carbohydrates, fatty acids, lipids, steroids,nucleotides, nucleotide analogues, nucleic acid molecules, nucleic acidanalogues, peptide nucleic acid molecules, small organic molecules, oreven polymers. The protein-based therapeutics of the present inventioncan also comprise modified or non-naturally occurring amino acids.Non-amino acid portions of the compounds can serve any purpose,including but not limited to: facilitating the purification of thecompound, improving the solubility or distribution or the compound (suchas in a therapeutic formulation), linking domains of the compound orlinking chemical moieties to the compound, contributing to thetwo-dimensional or three-dimensional structure of the compound,increasing the overall size of the compound, increasing the stability ofthe compound, and contributing to the anchoring activity or therapeuticactivity of the compound.

The peptide or protein-based compounds of the present invention can alsoinclude protein or peptide sequences in addition to those that compriseanchoring domains or therapeutic domains. The additional proteinsequences can serve any purpose, including but not limited to any of thepurposes outlined above (facilitating the purification of the compound,improving the solubility or distribution or the compound, linkingdomains of the compound or linking chemical moieties to the compound,contributing to the two-dimensional or three-dimensional structure ofthe compound, increasing the overall size of the compound, increasingthe stability of the compound, or contributing to the anchoring activityor therapeutic activity of the compound). Any additional protein oramino acid sequences can be part of a single polypeptide or proteinchain that includes the anchoring domain or domains and therapeuticdomain or domains, but any feasible arrangement of protein sequences iswithin the scope of the present invention.

The anchoring domain and therapeutic domain can be arranged in anyappropriate way that allows the compound to bind at or near a targetcell membrane. The compound can have at least one protein orpeptide-based anchoring domain and at least one peptide or protein-basedtherapeutic domain. In this case, the domains can be arranged linearlyalong the peptide backbone in any order. The anchoring domain can beN-terminal to the therapeutic domain, or can be C-terminal to thetherapeutic domain. It is also possible to have one or more therapeuticdomains flanked by at least one anchoring domain on each end.Alternatively, one or more anchoring domains can be flanked by at leastone therapeutic domain on each end. Chemical or peptide linkers canoptionally be used to join some or all of the domains of a compound.

It is also possible to have the domains in a nonlinear, branchedarrangement. For example, the therapeutic domain can be attached to aderivatized side chain of an amino acid that is part of a polypeptidechain that also includes, or is linked to, the anchoring domain.

A compound of the present invention can have more than one anchoringdomain. In cases in which a compound has more than one anchoring domain,the anchoring domains can be the same or different. A compound used inthe present invention can have more than one therapeutic domain. Incases in which a compound has more than one therapeutic domain, thetherapeutic domains can be the same or different. Where a compoundcomprises multiple anchoring domains, the anchoring domains can bearranged in tandem (with or without linkers) or on alternate sides ofother domains, such as therapeutic domains. Where a compound comprisesmultiple therapeutic domains, the therapeutic domains can be arranged intandem (with or without linkers) or on alternate sides of other domains,such as, but not limited to, anchoring domains.

A peptide or protein-based compound of the present invention can be madeby any appropriate way, including purifying naturally occurringproteins, optionally proteolytically cleaving the proteins to obtain thedesired functional domains, and conjugating the functional domains toother functional domains. Peptides can also be chemically synthesized,and optionally chemically conjugated to other peptides or chemicalmoieties. A peptide or protein-based compound of the present inventioncan be made by engineering a nucleic acid construct to encode at leastone anchoring domain and at least one therapeutic domain together (withor without nucleic acid linkers) in a continuous polypeptide. Thenucleic acid constructs, in some embodiments having appropriateexpression sequences, can be transfected into prokaryotic or eukaryoticcells, and the therapeutic protein-based compound can be expressed bythe cells and purified. Any desired chemical moieties can optionally beconjugated to the peptide or protein-based compound after purification.In some cases, cell lines can be chosen for expressing the protein-basedtherapeutic for their ability to perform desirable post-translationalmodifications (such as, but not limited to glycosylation).

A great variety of constructs can be designed and their protein productstested for desirable activities (such as, for example, binding activityof an anchoring domain, or a binding, catalytic, or inhibitory activityof a therapeutic domain).

Anchoring Domain

As used herein, an “extracellular anchoring domain” or “anchoringdomain” is any moiety that can stably bind an entity that is at or onthe exterior surface of a target cell or is in close proximity to theexterior surface of a target cell. An anchoring domain serves to retaina compound used in the present invention at or near the external surfaceof a target cell.

An extracellular anchoring domain can bind 1) a molecule expressed onthe surface of a target cell, or a moiety, domain, or epitope of amolecule expressed on the surface of a target cell, 2) a chemical entityattached to a molecule expressed on the surface of a target cell, or 3)a molecule of the extracellular matrix surrounding a target cell.

An anchoring domain can be a peptide or protein domain (including amodified or derivatized peptide or protein domain), or comprises amoiety coupled to a peptide or protein. A moiety coupled to a peptide orprotein can be any type of molecule that can contribute to the bindingof the anchoring domain to an entity at or near the target cell surface,and in some embodiments is an organic molecule, such as, for example,nucleic acid, peptide nucleic acid, nucleic acid analogue, nucleotide,nucleotide analogue, small organic molecule, polymer, lipids, steroid,fatty acid, carbohydrate, or any combination of any of these.

A molecule, complex, domain, or epitope that is bound by an anchoringdomain may or may not be specific for the target cell. For example, ananchoring domain may bind an epitope present on molecules on or in closeproximity to the target cell and that occur at sites other than thevicinity of the target cell as well. In many cases, however, localizeddelivery of a therapeutic compound of the present invention willrestrict its occurrence primarily to the surface of target cells. Inother cases, a molecule, complex, moiety, domain, or epitope bound by ananchoring domain may be specific to a target tissue or target cell type.

Target tissue or target cell type includes the sites in an animal orhuman body where a pathogen invades or amplifies. For example, a targetcell can be an endothelial cell that can be infected by a pathogen. Acomposition used in the present invention can comprise an anchoringdomain that can bind a cell surface epitope, for example, that isspecific for the endothelial cell type. In another example, a targetcell can be an epithelial cell and a composition of the presentinvention can bind an epitope present on the cell surface of manyepithelial cell types, or present in the extracellular matrix ofdifferent types of epithelial cells. In this case localized delivery ofthe composition can restrict its localization to the site of theepithelial cells that are targets of the pathogen.

Compounds used in the present invention can have one or more anchoringdomains that can bind at or near the surface of epithelial cells. Forexample, heparan sulfate, closely related to heparin, is a type ofglycosaminoglycan (GAG) that is ubiquitously present on cell membranes,including the surface of respiratory epithelium. Many proteinsspecifically bind to heparin/heparan sulfate, and the GAG-bindingsequences in these proteins have been identified (Meyer, F A, King, Mand Gelman, R A. (1975) Biochimica et Biophysica Acta 392: 223-232;Schauer, S. ed., pp 233. Sialic Acids Chemistry, Metabolism andFunction. Springer-Verlag, 1982). For example, the GAG-binding sequencesof human platelet factor 4 (PF4) (SEQ ID NO:2), human interleukin 8(IL8) (SEQ ID NO:3), human antithrombin III (AT III) (SEQ ID NO:4),human apoprotein E (ApoE) (SEQ ID NO:5), human angio-associatedmigratory cell protein (AAMP) (SEQ ID NO:6), or human amphiregulin (SEQID NO:7) (FIG. 1) have been shown to have very high affinity (in thenanomolar range) towards heparin (Lee, M K and Lander, A D. (1991) ProNatl Acad Sci USA 88:2768-2772; Goger, B, Halden, Y, Rek, A, Mosl, R,Pye, D. Gallagher, J and Kungl, A J. (2002) Biochem. 41:1640-1646; Witt,D P and Lander A D (1994) Curr Bio 4:394-400; Weisgraber, K H, Rall, SC, Mahley, R W, Milne, R W and Marcel, Y. (1986) J Bio Chem261:2068-2076). The GAG-binding sequences of these proteins are distinctfrom their receptor-binding sequences, so they will not induce thebiological activities associated with the full-length proteins or thereceptor-binding domains. These sequences, or other sequences that havebeen identified or are identified in the future as heparin/heparansulfate binding sequences, or sequences substantially homologous toidentified heparin/heparan sulfate binding sequences that haveheparin/heparan sulfate binding activity, can be used asepithelium-anchoring-domains in compounds used in the present invention.

An anchoring domain can bind a moiety that is specific to the targetcell type of a particular species or can bind a moiety that is found inthe target cell type of more than one species.

Therapeutic Domain

A compound used in the present invention includes at least onetherapeutic domain or active portion, those terms being usedinterchangeable herein. The therapeutic activity can be, as nonlimitingexamples, a binding activity, a catalytic activity, or an inhibitoryactivity. A therapeutic domain can modify or inhibit a function of thetarget cell or target organism. An active portion of a compound hastherapeutic activity. For example, the catalytic domain or activeportion of a sialidase can be its therapeutic domain.

The therapeutic domain can act extracellularly, meaning that itsinfection-preventing, inflammatory response-modulating, ortransduction-enhancing activity takes place at the target cell surfaceor in the immediate area surrounding the target cell, including siteswithin the extracellular matrix, intracellular spaces, or luminal spacesof tissues.

A therapeutic domain can be a peptide or protein domain (including amodified or derivatized peptide or protein domain), or comprises amoiety coupled to a peptide or protein. A moiety coupled to a peptide orprotein can be any type of molecule, and is in some embodiments anorganic molecule, such as, for example, nucleic acid, peptide nucleicacid, nucleic acid analogue, nucleotide, nucleotide analogue, smallorganic molecule, polymer, lipids, steroid, fatty acid, carbohydrate, orany combination of any of these.

A therapeutic domain can be a synthetic peptide or polypeptide, or cancomprise a synthetic molecule that can be conjugated to a peptide orpolypeptide, can be a naturally-occurring peptide or protein, or adomain of naturally-occurring protein. A therapeutic domain can also bea peptide or protein that is substantially homologous to anaturally-occurring peptide or protein.

Linkers

A compound used in the present invention can optionally include one ormore linkers that can join domains of the compound. Linkers can be usedto provide optimal spacing or folding of the domains of a compound. Thedomains of a compound joined by linkers can be therapeutic domains,anchoring domains, or any other domains or moieties of the compound thatprovide additional functions such as enhancing compound stability,facilitating purification, etc. A linker used to join domains ofcompounds of the present invention can be a chemical linker or an aminoacid or peptide linker. Where a compound comprises more than one linker,the linkers can be the same or different. Where a compound comprisesmore than one linker, the linkers can be of the same or differentlengths.

Many chemical linkers of various compositions, polarity, reactivity,length, flexibility, and cleavability are known in the art of organicchemistry. Preferred linkers include amino acid or peptide linkers.Peptide linkers are well known in the art. Some embodiments of linkersare between one and about one hundred amino acids in length, and betweenone and about thirty amino acids in length, although length is not alimitation in the linkers of the compounds of the present invention. Thelinker amino acid sequences can be selected such that they do notinterfere with the mucus-reducing and/or anti-inflammatory activity ofthe compounds and compositions used in the present invention. Someembodiments of linkers are those that include the amino acid glycine.For example, linkers having the sequence:

(GGGGS (SEQ ID NO:10))n, where n is a whole number between 1 and 20, orbetween 1 and 12, can be used to link domains of therapeutic compoundsused in the present invention.Composition Comprising at least one Anchoring Domain and at least oneCatalytic Activity

In some aspects, the present invention can use compounds that have atherapeutic domain that has an enzymatic activity. The enzymaticactivity can be a catalytic activity that removes, degrades or modifiesa host molecule or complex. In some embodiments the host molecule orcomplex can be removed, degraded, or modified by the enzymatic activityof a compound of the present invention is on, at, or near the surface ofa target cell.

Compounds used in the present invention can have, for example, one ofthe following structures:

(Anchoring Domain)n-[linker]-(Enzymatic Activity)n (n=1,2, 3 or more)

-   -   or :

(Enzymatic Activity)n (n=1,2, 3 or more)-[linker]-(Anchoring Domain)n,

-   -   where the linkers are optional.

The enzymatic activity can be a monomeric form of a peptide orpolypeptide or can be multiple copies of the same polypeptide that areeither linked directly or with spacing sequence in between. Thepolypeptides or peptides can be linked directly or via a spacer composedof peptide linker sequence. The anchoring domain can be any peptide orpolypeptide that can bind to or near the surface of target cells.

In one embodiment, a therapeutic domain comprises a sialidase that caneliminate or greatly reduce the level of sialic acid on the surface ofepithelial cells. The therapeutic domain can comprise a completesialidase protein, or an active portion thereof, wherein the activeportion thereof retains the ability to perform the catalytic function(s)of the sialidase protein (e.g., cleaving sialic acid residues).

Sialic acid mediates cell adhesion and interactions between inflammatorycells and target cells. Therefore, treating the surface of respiratoryepithelial cells with a sialidase can prevent the recruitment ofinflammatory cells to the airway surface, and therefore can treatallergic reactions including asthma and allergic rhinitis. It alsounexpectedly results in reducing the quantity of mucus in therespiratory tract of subjects with elevated levels of mucus in theirrespiratory tract, and limiting increase in the quantity of mucus in therespiratory tract of subjects above a baseline of mucus in therespiratory tract of those subjects.

Among the sialidases contemplated for use in the methods describedherein are the large bacterial sialidases that can degrade the receptorsialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. Forexample, the bacterial sialidase enzymes from Clostridium perfringens(Genbank Accession Number X87369), Actinomyces viscosus, Arthrobacterureafaciens, or Micromonospora viridifaciens (Genbank Accession NumberD01045) can be used. Therapeutic domains of compounds of the presentinvention can comprise all or a portion of the amino acid sequence of alarge bacterial sialidase or can comprise amino acid sequences that aresubstantially homologous to all or a portion of the amino acid sequenceof a large bacterial sialidase. In one preferred embodiment, atherapeutic domain comprises a sialidase encoded by Actinomycesviscosus, such as that of SEQ ID NO:12, or such as sialidase sequencesubstantially homologous to SEQ ID NO:12. In yet another preferredembodiment, a therapeutic domain comprises the catalytic domain of theActinomyces viscosus sialidase extending from amino acids 274-667 of SEQID NO:12, or a substantially homologous sequence.

Other sialidases contemplated for use in the methods described hereinare the human sialidases such as those encoded by the genes NEU2 (SEQ IDNO:8; Genbank Accession Number Y16535; Monti, E, Preti, Rossi, E.,Ballabio, A and Borsani G. (1999) Genomics 57:137-143) and NEU4 (SEQ IDNO:9; Genbank Accession Number NM080741; Monti, E, Preti, A, Venerando,B and Borsani, G. (2002) Neurochem Res 27:646-663) (FIG. 2). Therapeuticdomains of compounds used in the present invention can comprise all or aportion of the amino acid sequences of a human sialidase or can compriseamino acid sequences that are substantially homologous to all or aportion of the amino acid sequences of a human sialidase. Where atherapeutic domain comprises a portion of the amino acid sequences of anaturally occurring sialidase, or sequences substantially homologous toa portion of the amino acid sequences of a naturally occurringsialidase, the portion can have essentially the same activity as thehuman sialidase.

A compound for reducing elevated levels of mucus in the respiratorytract can in some embodiments have one or anchoring domains that canbind at or near the surface of epithelial cells. In some embodiments,the epithelium-anchoring domain is a GAG-binding sequence from a humanprotein, such as, for example, the GAG-binding amino acid sequences ofhuman platelet factor 4 (PF4) (SEQ ID NO:2), human interleukin 8 (IL8)(SEQ ID NO:3), human antithrombin III (AT III) (SEQ ID NO:4), humanapoprotein E (ApoE) (SEQ ID NO:5), human angio-associated migratory cellprotein (AAMP) (SEQ ID NO:6), and human amphiregulin (SEQ ID NO:7) (FIG.1). An epithelial anchoring domain can also be substantially homologousto a naturally occurring GAG-binding sequence, such as those listed inFIG. 1. Such compounds can be formulated for nasal, tracheal, bronchial,oral, or topical administration, or can be formulated as an injectablesolution or as eyedrops, or formulated into a solution or dry powder andinhaled with inhalers.

A pharmaceutical composition comprising such compounds can be used totreat or prevent allergy or inflammatory response. In addition, suchcompounds have been shown herein to reduce the quantity of mucus in therespiratory tract of subjects with elevated levels of mucus in theirrespiratory tracts, and to limit increases in the quantity of mucus inthe respiratory tract of subjects above a baseline of mucus in theirrespiratory tracts. Therefore, such compounds can be used to astherapeutic treatments to reduce the quantity of mucus in therespiratory tract of subjects with elevated levels of mucus in theirrespiratory tracts, or as prophylactic treatments to limit increases inthe quantity of mucus in the respiratory tract of subjects above abaseline of mucus in their respiratory tracts. Due to their effect onmucus in the respiratory tract, these compounds can also be used toprevent, treat, or ameliorate the effects of chronic obstructivepulmonary disease (COPD), bronchitis, bronchiectasis, cystic fibrosis(CF), vasculitis, mucus plugging, Wegener's granulomatosis, pneumonia,tuberculosis, cancer involving the lungs or the respiratory tract,Kartagener syndrome, Young's syndrome, chronic sinopulmonary infection,alpha 1-antitrypsin deficiency, primary immunodeficiency, acquiredimmune deficiency syndrome, opportunistic infection, an infectiousstate, a post infectious state, common cold, exercise inducedhypersecretion of mucus, inflammatory bowel disease, ulcerative colitis,Crohn's disease, respiratory infection, respiratory obstruction,inhalation or aspiration of a toxic gas, pulmonary aspiration, oralcoholism in subjects with elevated levels of mucus in theirrespiratory tract or who are at risk of having increased levels of mucusin their respiratory tract.

It is also within the scope of the present invention to use compounds orcompositions comprising a human sialidase, such as any of thosedescribed herein, or an active portion thereof, or a compound withsubstantial homology to a sialidase, in the absence of an anchoringdomain (a) to treat or prevent allergic and inflammatory responses inthe respiratory tract, (b) to reduce the quantity of mucus in therespiratory tract of subjects with elevated levels of mucus in theirrespiratory tracts, (c) to limit increases in the quantity of mucus inthe respiratory tract of subjects above a baseline of mucus in theirrespiratory tracts, and/or (d) to prevent, treat, or ameliorate theeffects of chronic obstructive pulmonary disease (COPD), bronchitis,bronchiectasis, cystic fibrosis (CF), vasculitis, mucus plugging,Wegener's granulomatosis, pneumonia, tuberculosis, cancer involving thelungs or the respiratory tract, Kartagener syndrome, Young's syndrome,chronic sinopulmonary infection, alpha 1-antitrypsin deficiency, primaryimmunodeficiency, acquired immune deficiency syndrome, opportunisticinfection, an infectious state, a post infectious state, common cold,exercise induced hypersecretion of mucus, inflammatory bowel disease,ulcerative colitis, Crohn's disease, respiratory infection, respiratoryobstruction, inhalation or aspiration of a toxic gas, pulmonaryaspiration, or alcoholism in subjects with elevated levels of mucus intheir respiratory tract or who are at risk of having increased levels ofmucus in their respiratory tract. The present invention recognizes thatelevated levels of mucus in the respiratory tract can be reduced by theuse of a sialidase or an active portion of a sialidase, and that suchsialidases or active portions thereof can optionally be adapted, bygenetic or chemical engineering, or by pharmaceutical formulation, toimprove their half life or retention at the respiratory epithelium.

These compounds and pharmaceutical compositions can be delivered to theupper respiratory tract as a nasal spray, or delivered to therespiratory tract as an inhalant with inhalers.

The compounds described herein can be formulated into pharmaceuticalcompositions that include various additional compounds either alone orin various combinations, such as, Na₂SO₄, MgSO₄, CaCl_(2,) Histidine,Histine-HCl, and Trehalose or their analogs. These additional compoundscan be included in the pharmaceutical compositions to act as excipientsor as active ingredients that provide additional beneficial effects.

Therapeutic Composition Comprising at least one Sialidase Activity

The present invention includes methods that use therapeutic compoundsand compositions that comprise at least one sialidase activity. Thesialidase activity can be a sialidase isolated from any source, such as,for example, a bacterial or mammalian source, or can be a recombinantprotein that is substantially homologous to at least a portion of anaturally occurring sialidase. In some embodiments sialidases are thelarge bacterial sialidases that can degrade the receptor sialic acidsNeu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For example, thebacterial sialidase enzymes from Clostridium perfringens (GenbankAccession Number X87369), Actinomyces viscosus (Genbank Accession NumberL06898), Arthrobacter ureafaciens, or Micromonospora viridifaciens(Genbank Accession Number D01045) or substantially homologous proteinscan be used.

For example, therapeutic compounds and compositions used in the presentinvention can comprise a large bacterial sialidase or can comprise aprotein with the amino acid sequence of a large bacterial sialidase orcan comprise amino acid sequences that are substantially homologous tothe amino acid sequence of a large bacterial sialidase. A pharmaceuticalcomposition that can be used in the present invention comprises the A.viscosus sialidase (SEQ ID NO:12), or comprises a protein substantiallyhomologous to the A. viscosus sialidase.

Other sialidases that can be used in the compositions, compounds andmethods described herein are the human sialidases such as those encodedby the genes NEU2 (SEQ ID NO:8; Genbank Accession Number Y16535; Monti,E, Preti, Rossi, E., Ballabio, A and Borsani G. (1999) Genomics57:137-143) and NEU4 (SEQ ID NO:9; Genbank Accession Number NM080741;Monti, E, Preti, A, Venerando, B and Borsani, G. (2002) Neurochem Res27:646-663) (FIG. 2). Therapeutic domains of compounds of the presentinvention can comprise a human sialidase protein that is substantiallyhomologous to the amino acid sequences of a human sialidase or cancomprise amino acid sequences that are substantially homologous to allor a portion of the amino acid sequences of a human sialidase. Where atherapeutic domain comprises a portion of the amino acid sequences of anaturally occurring sialidase, or sequences substantially homologous toa portion of the amino acid sequences of a naturally occurringsialidase, the portion can have essentially the same activity as thehuman sialidase, e.g., an active portion of the sialidase.

Generally, sialidases that can effectively degrade on respiratoryepithelial cells both receptor sialic acids Neu5Ac α(2,6)-Gal and Neu5Acα(2,3)-Gal, can be used. Sialidases are found in higher eukaryotes, aswell as in some mostly pathogenic microbes, including viruses, bacteriaand protozoans. Viral and bacterial sialidases have been wellcharacterized, and the three-dimensional structures of some of them havebeen determined (Crennell, S J, Garman, E, Laver, G, Vimr, E and Taylor,G. (1994) Structure 2:535-544; Janakiraman, M N, White, C L, Laver, W G,Air, G M and Luo, M. (1994) Biochemistry 33:8172-8179; Pshezhetsky, A,Richard, C, Michaud, L, Igdoura, S, Wang, S, Elsliger, M, Qu, J,Leclerc, D, Gravel, R, Dallaire, L and Potier, M. (1997) Nature Genet15: 316-320). Several human sialidases have also been cloned in therecent years (Milner, C M, Smith, S V, Carrillo M B, Taylor, G L,Hollinshead, M and Campbell, R D. (1997) J Bio Chem 272:4549-4558;Monti, E, Preti, A, Nesti, C, Ballabio, A and Borsani G. 1999. Glycobiol9:1313-1321; Wada, T, Yoshikawa, Y, Tokuyama, S, Kuwabara, M, Akita, Hand Miyagi, T. (1999) Biochem Biophy Res Communi 261:21-27; Monti, E,Bassi, M T, Papini, N, Riboni, M, Manzoni, M, Veneranodo, B, Croci, G,Preti, A, Ballabio, A, Tettamanti, G and Borsani, G. (2000) Bichem J349:343-351). DAS181, which contains an active portion of a sialidase,has also been cloned.

All the sialidases characterized share a four amino acid motif in theamino terminal portion followed by the Asp box motif which is repeatedthree to five times depending on the protein. (Monti, E, Bassi, M T,Papini, N, Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti, A,Ballabio, A, Tettamanti, G and Borsani, G. (2000) Bichem J 349:343-351;Copley, R R, Russell, R B and Ponting, C P. (2001) Protein Sci10:285-292). While the overall amino acid identity of the sialidasesuperfamily is relatively low at about 20-30%, the overall fold of themolecules, especially the catalytic amino acids, are remarkably similar(Wada, T, Yoshikawa, Y, Tokuyama, S, Kuwabara, M, Akita, H and Miyagi,T. (1999) Biochem Biophy Res Communi 261:21-27; Monti, E, Bassi, M T,Papini, N, Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti, A,Ballabio, A, Tettamanti, G and Borsani, G. (2000) Bichem J 349:343-351;Copley, R R, Russell, R B and Ponting, C P. (2001) Protein Sci10:285-292).

The sialidases are generally divided into two families: “small”sialidases have molecular weight of about 42 kDa and do not requiredivalent metal ion for maximal activity; “large” sialidases havemolecular weight above 65 kDa and may require divalent metal ion foractivity (Wada, T, Yoshikawa, Y, Tokuyama, S, Kuwabara, M, Akita, H andMiyagi, T. (1999) Biochem Biophy Res Communi 261:21-27; Monti, E, Bassi,M T, Papini, N, Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti,A, Ballabio, A, Tettamanti, G and Borsani, G. (2000) Bichem J349:343-351; Copley, R R, Russell, R B and Ponting, C P. (2001) ProteinSci 10:285-292).

Over fifteen sialidase proteins have been purified and they vary greatlyfrom one another in substrate specificities and enzymatic kinetics.Large bacterial sialidases can effectively cleave sialic acid in both(α,2-6) linkage and (α,2-3) linkage in the context of most naturalsubstrates (FIG. 4; Vimr, D R. (1994) Trends Microbiol 2: 271-277;Drzeniek, R. (1973) Histochem J 5:271-290; Roggentin, P, Kleineidam, R Gand Schauer, R. (1995) Biol Chem Hoppe-Seyler 376:569-575; Roggentin, P,Schauer, R, Hoyer, L L and Vimr, E R. (1993) Mol Microb 9:915-921).Because of their broad substrate specificities, large bacterialsialidases make good candidates.

FIG. 4 shows several of the large bacterial sialidases with knownsubstrate specificity. These enzymes have high specific activity (600U/mg protein for C. perfringens (Corfield, A P, Veh, R W, Wember, M,Michalski, J C and Schauer, R. (1981) Bichem J 197:293-299) and 680 U/mgprotein for A. viscosus (Teufel, M, Roggentin, P. and Schauer, R. (1989)Biol Chem Hoppe Seyler 370:435-443)), are fully active without divalentmetal iron, and have been cloned and purified as recombinant proteinsfrom E. coli (Roggentin, P, Kleineidam, R G and Schauer, R. (1995) BiolChem Hoppe-Seyler 376:569-575, Teufel, M, Roggentin, P. and Schauer, R.(1989) Biol Chem Hoppe Seyler 370:435-443 , Sakurada, K, Ohta, T andHasegawa, M. (1992) J Bacteriol 174: 6896-6903). In addition, C.perfringens is stable in solution at 2-8° C. for several weeks, and at4° C. in the presence of albumin for more than two years (Wang, F Z,Akula, S M, Pramod, N P, Zeng, L and Chandran, B. (2001) J Virol75:7517-27). A. viscosus is labile towards freezing and thawing, but isstable at 4° C. in 0.1 M acetate buffer, pH 5 (Teufel, M, Roggentin, P.and Schauer, R. (1989) Biol Chem Hoppe Seyler 370:435-443).

A pharmaceutical composition comprising a sialidase can include othercompounds, including but not limited to other proteins, that can alsohave therapeutic activity. A pharmaceutical composition comprising asialidase can include other compounds that can enhance the stability,solubility, packaging, delivery, consistency, taste, or fragrance of thecomposition.

Compounds comprising a sialidase can be formulated for nasal, tracheal,bronchial, oral, or topical administration, or can be formulated as aninjectable solution or as eyedrops, or formulated into a solution or drypowder and inhaled with inhalers. The sialidases described herein can beformulated into pharmaceutical compositions that include variousadditional compounds such as, MgSO₄, CaCl₂, Histidine, Histine-HCl, andTrehalose or their analogs.

These sialidases or pharmaceutical compositions containing them can beused (a) to treat or prevent allergic and inflammatory responses in therespiratory tract, (b) to reduce the quantity of mucus in therespiratory tract of subjects with elevated levels of mucus in theirrespiratory tracts, (c) to limit increases in the quantity of mucus inthe respiratory tract of subjects above a baseline of mucus in theirrespiratory tracts, and/or (d) to prevent, treat, or ameliorate theeffects of chronic obstructive pulmonary disease (COPD), bronchitis,bronchiectasis, cystic fibrosis (CF), vasculitis, mucus plugging,Wegener's granulomatosis, pneumonia, tuberculosis, cancer involving thelungs or the respiratory tract, Kartagener syndrome, Young's syndrome,chronic sinopulmonary infection, alpha 1-antitrypsin deficiency, primaryimmunodeficiency, acquired immune deficiency syndrome, opportunisticinfection, an infectious state, a post infectious state, common cold,exercise induced hypersecretion of mucus, inflammatory bowel disease,ulcerative colitis, Crohn's disease, respiratory infection, respiratoryobstruction, inhalation or aspiration of a toxic gas, pulmonaryaspiration, or alcoholism in subjects with elevated levels of mucus intheir respiratory tract or who are at risk of having increased levels ofmucus in their respiratory tract. In some embodiments, subjects withelevated levels of mucus in their respiratory tract do not includesubjects with one or more of influenza, parainfluenza, and/orrespiratory syncytial virus (RSV).

Sialidase Catalytic Domain Proteins or Peptides

As used herein a “sialidase catalytic domain protein or peptide”comprises a catalytic domain of a sialidase but does not comprise theentire amino acid sequence of the sialidase from which the catalyticdomain is derived. A sialidase catalytic domain protein or peptide hassialidase activity. A sialidase catalytic domain protein or peptide canhave at least 10%, at least 20%, at least 50%, at least 70% of theactivity of the sialidase from which the catalytic domain sequence isderived. A sialidase catalytic domain protein or peptide can have atleast 90% of the activity of the sialidase from which the catalyticdomain sequence is derived.

A sialidase catalytic domain protein or peptide can include other aminoacid sequences, such as but not limited to additional sialidasesequences, sequences derived from other proteins, or sequences that arenot derived from sequences of naturally-occurring proteins. Additionalamino acid sequences can perform any of a number of functions, includingcontributing other activities to the catalytic domain protein, enhancingthe expression, processing, folding, or stability of the sialidasecatalytic domain protein, or even providing a desirable size or spacingof the protein or peptide.

A preferred sialidase catalytic domain protein or peptide is a proteinthat comprises the catalytic domain of the A. viscosus sialidase. An A.viscosus sialidase catalytic domain protein or peptide can include aminoacids 270-667 of the A. viscosus sialidase sequence (SEQ ID NO:12). AnA. viscosus sialidase catalytic domain protein or peptide can includeamino acid sequence that begins at any of the amino acids from aminoacid 270 to amino acid 290 of the A. viscosus sialidase sequence (SEQ IDNO:12) and ends at any of the amino acids from amino acid 665 to aminoacid 901 of said A. viscosus sialidase sequence (SEQ ID NO:12), andlacks any A. viscosus sialidase protein sequence extending from aminoacid 1 to amino acid 269. (As used herein “lacks any A. viscosussialidase protein sequence extending from amino acid 1 to amino acid269” means lacks any stretch of four or more consecutive amino acids asthey appear in the designated protein or amino acid sequence.)

In some embodiments, an A. viscosus sialidase catalytic domain proteinor peptide comprises amino acids 274-681 of the A. viscosus sialidasesequence (SEQ ID NO:12) and lacks other A. viscosus sialidase sequence.In other embodiments, an A. viscosus sialidase catalytic domain proteincomprises amino acids 290-666 or 290-667 of the A. viscosus sialidasesequence (SEQ ID NO:12) and lacks any other A. viscosus sialidasesequence. In yet other embodiments, an A. viscosus sialidase catalyticdomain protein or peptide comprises amino acids 274-666 of the A.viscosus sialidase sequence (SEQ ID NO:12) and lacks any other A.viscosus sialidase sequence. In yet other embodiments, an A. viscosussialidase catalytic domain protein or peptide comprises amino acids290-666 or 290-667 of the A. viscosus sialidase sequence (SEQ ID NO:12)and lacks any other A. viscosus sialidase sequence. In yet otherembodiments, an A. viscosus sialidase catalytic domain protein orpeptide comprises amino acids 290-681 of the A. viscosus sialidasesequence (SEQ ID NO:12) and lacks any other A. viscosus sialidasesequence.

Such sialidase catalytic domain proteins or peptides can be formulatedfor nasal, tracheal, bronchial, oral, or topical administration, or canbe formulated as an injectable solution or as eyedrops, or formulatedinto a solution or dry powder and inhaled with an inhaler. The sialidasecatalytic domain proteins or peptides described herein can be formulatedinto pharmaceutical compositions that include various additionalcompounds, such as, MgSO₄, CaCl₂, Histidine, Histine-HCl, and Trehaloseor their analogs. These additional compounds can be included in thepharmaceutical compositions either alone or in various combinations,such as, Na₂SO₄, MgSO₄, CaCl₂, Histidine, Histine-HCl, and Trehalose ortheir analogs. These additional compounds can be included in thepharmaceutical compositions to act as excipients or as activeingredients that provide additional beneficial effects.

Such sialidase catalytic domain proteins or peptides or pharmaceuticalcompositions containing them can be used (a) to treat or preventallergic and inflammatory responses in the respiratory tract, (b) toreduce the quantity of mucus in the respiratory tract of subjects withelevated levels of mucus in their respiratory tracts, (c) to limitincreases in the quantity of mucus in the respiratory tract of subjectsabove a baseline of mucus in their respiratory tracts, and/or (d) toprevent, treat, or ameliorate the effects of chronic obstructivepulmonary disease (COPD), bronchitis, bronchiectasis, cystic fibrosis(CF), vasculitis, mucus plugging, Wegener's granulomatosis, pneumonia,tuberculosis, cancer involving the lungs or the respiratory tract,Kartagener syndrome, Young's syndrome, chronic sinopulmonary infection,alpha 1-antitrypsin deficiency, primary immunodeficiency, acquiredimmune deficiency syndrome, opportunistic infection, an infectiousstate, a post infectious state, common cold, exercise inducedhypersecretion of mucus, inflammatory bowel disease, ulcerative colitis,Crohn's disease, respiratory infection, respiratory obstruction,inhalation or aspiration of a toxic gas, pulmonary aspiration, oralcoholism in subjects with elevated levels of mucus in theirrespiratory tract or who are at risk of having increased levels of mucusin their respiratory tract.

Fusion Proteins

Sialidase catalytic domain proteins can be fusion proteins, in which thefusion protein comprises at least one sialidase catalytic domain and atleast one other protein domain, including but not limited to: apurification domain, a protein tag, a protein stability domain, asolubility domain, a protein size-increasing domain, a protein foldingdomain, a protein localization domain, an anchoring domain, anN-terminal domain, a C-terminal domain, a catalytic activity domain, abinding domain, or a catalytic activity-enhancing domain. The at leastone other protein domain can be derived from another source, such as,but not limited to, sequences from another protein. The at least oneother protein domain need not be based on any known protein sequence,but can be engineered and empirically tested to perform any function inthe fusion protein.

Purification domains can include, as nonlimiting examples, one or moreof a his tag, a calmodulin binding domain, a maltose binding proteindomain, a streptavidin domain, a streptavidin binding domain, an inteindomain, or a chitin binding domain. Protein tags can comprise sequencesthat can be used for antibody detection of proteins, such as, forexample, the myc tag, the hemagglutinin tag, or the FLAG tag. Proteindomains that enhance protein expression, modification, folding,stability, size, or localization can be based on sequences of knowproteins or engineered. Other protein domains can have binding orcatalytic activity or enhance the catalytic activity of the sialidasecatalytic domain.

Fusion proteins used in the compositions, compounds and methods of thepresent invention comprise at least one sialidase catalytic domain andat least one anchoring domain. In some embodiments, anchoring domainsinclude GAG-binding domains, such as the GAG-binding domain or humanamphiregulin (SEQ ID NO:7).

Sialidase catalytic domains and other domains of a fusion protein usedin the present invention can optionally be joined by linkers, such asbut not limited to peptide linkers. A variety of peptide linkers areknown in the art. In one embodiment a linker can be a peptide linkercomprising glycine, such as G-G-G-G-S (SEQ ID NO:10).

Such fusion proteins can be formulated for nasal, tracheal, bronchial,oral, or topical administration, or can be formulated as an injectablesolution or as eyedrops or formulated into a solution or dry powder andinhaled with an inhaler. These fusion proteins can be formulated intopharmaceutical compositions that include various additional compoundseither alone or in various combinations, such as, Na₂SO₄, MgSO₄, CaCl₂,Histidine, Histine-HCl, and Trehalose or their analogs. These additionalcompounds can be included in the pharmaceutical compositions to act asexcipients or as active ingredients that provide additional beneficialeffects.

Such fusion proteins or pharmaceutical compositions containing them canbe used (a) to treat or prevent allergic and inflammatory responses inthe respiratory tract, (b) to reduce the quantity of mucus in therespiratory tract of subjects with elevated levels of mucus in theirrespiratory tracts, (c) to limit increases in the quantity of mucus inthe respiratory tract of subjects above a baseline of mucus in theirrespiratory tracts, and/or (d) to prevent, treat, or ameliorate theeffects of chronic obstructive pulmonary disease (COPD), bronchitis,bronchiectasis, cystic fibrosis (CF), vasculitis, mucus plugging,Wegener's granulomatosis, pneumonia, tuberculosis, cancer involving thelungs or the respiratory tract, Kartagener syndrome, Young's syndrome,chronic sinopulmonary infection, alpha 1-antitrypsin deficiency, primaryimmunodeficiency, acquired immune deficiency syndrome, opportunisticinfection, an infectious state, a post infectious state, common cold,exercise induced hypersecretion of mucus, inflammatory bowel disease,ulcerative colitis, Crohn's disease, respiratory infection, respiratoryobstruction, inhalation or aspiration of a toxic gas, pulmonaryaspiration, or alcoholism in subjects with elevated levels of mucus intheir respiratory tract or who are at risk of having increased levels ofmucus in their respiratory tract.

Various constructs of fusion proteins are shown in FIGS. 4-6, as well asin the sequences provided in the sequence listing provided herein.

Methods for Testing the Compounds and Compositions and/or for Screeningto Identify Sialidases and/or Active Portions Thereof to Treat DiseasesAccompanied by Inflammation

The compounds and compositions provided herein can be tested for theiractivity in reducing inflammation, allergies or associated responses,such as mucus overproduction, using standard assays known to those ofskill in the art. Several cell-based (e.g., tracheal cell cultures) andanimal-based assays (mouse models, guinea pig models) for measuringinflammation or mucus overproduction are known (see, e.g., Nakao et al.,J. Immunol., 180:6262-6269 (2008); Westerhof et al., Mediators Inflamm.,10(3):143-154 (2001); Miller et al., J. Immunol., 170:3348-3356 (2003);Nakanishi et al., Proc. Natl. Acad. Sci. USA, 98(9):5175-5180 (2001);and DuBuske, Allergy Proc., 16(2):55-58 (1995), the contents of each ofwhich are incorporated in their entirety by reference herein). Thecompounds and compositions provided herein can be tested for theirability to reduce inflammation or mucus overproduction in any of theseassays or other standard assays known to those of skill in the art. Inaddition, sialidases or active portions thereof can be identified and/orselected for their anti-inflammatory activity and/or ability to reduceassociated responses, such as mucus overproduction, using such assays.Exemplary assays and protocols are described herein in Example 1 andExample 2.

-   In addition to assays that measure inflammation or associated    responses, such as mucus overproduction, the compounds and    compositions provided herein can be tested for their activity by    assessing their ability to disrupt muscarinic receptor-mediated    signaling in the presence of an agonist. Muscarinic receptors, or    mAChRs, are G protein-coupled acetylcholine receptors found in the    plasma membranes of certain neurons-and other cells. They play    several roles, including acting as the main end-receptor stimulated    by acetylcholine released from postganglionic fibers in the    parasympathetic nervous system.-   Muscarinic receptor-agonist interactions, and the resulting    signaling, is believed to play a role in diseases that have    associated inflammatory and/or allergic responses, such as asthma    and COPD (see, e.g., “Muscarinic Receptors in Airways Diseases,”    Birkhauser-Verlag publ., Zangsma et al., Eds.).-   More specifically, acetylcholinergic mechanisms are recognized to    influence the following normal and pathogenic respiratory functions:-   1. secretion of mucus,-   2. active transport of ions across the respiratory epithelium and    during mucociliary transport,-   3. smooth muscle tone of the airways,-   4. immunologic and inflammatory response of the airways,-   5. reflex regulation of the airways,-   6. respiratory responses of the airways in asthma and in other    hypersensitivity states of the respiratory tract.    Consequently, certain anti-muscarinic agents have been effective    against: (a) acetylcholinergically induced bronchoconstriction; (b)    iatrogenic airway spasms induced by beta blockers; and (c)    psychogenic bronchospasm. The two main pulmonary applications of    anti-muscarinic agents has been chronic bronchitis and bronchial    asthma (Pharmacology of Anti-Muscarinic Agents, Laszlo Gyermek    (1998)).-   There are five broad classes of muscarinic receptors, based on their    physiological roles, and agonists for each of these receptors are    known to those of skill in the art:-   M1 receptor—exemplary agonists include acetylcholine, oxotremorine,    muscarine, carbachol and McNA343-   M2 receptor—exemplary agonists include acetylcholine, methacholine,    carbachol, oxotremorine and muscarine-   M3 receptor—exemplary agonists include acetylcholine, bethanechol,    carbachol, oxotremorine and pilocarpine-   M4 receptor—exemplary agonists include acetylcholine, carbachol and    oxotremorine-   M5 receptor—exemplary agonists include acetylcholine, carbachol and    oxotremorine-   In some embodiments, the compounds and compositions provided herein    can be tested for the ability to reduce inflammation and/or allergic    responses, including mucus overproduction, associated with RTIs or    RTDs by assessing their ability to disrupt muscarinic    receptor-agonist interactions. Further, sialidases and/or active    portions thereof can be screened, identified and selected for their    ability to reduce inflammation, allergies, and/or associated    responses such as mucus overproduction by assessing their ability to    disrupt muscarinic receptor-agonist interactions. These tests and    screens can be performed using standard assays known to those of    skill in the art (see, e.g, Armstrong et al., Curr. Protocols in    Pharmacol., UNIT 12-13 (2010), the contents of which are    incorporated in their entirety by reference herein). An exemplary    assay and protocol is provided herein in Example 3.

Pharmaceutical Compositions

The present invention includes compounds of the present inventionformulated as pharmaceutical compositions. The pharmaceuticalcompositions comprise a pharmaceutically acceptable carrier prepared forstorage and subsequent administration, which have a pharmaceuticallyeffective amount of the compound in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, 18th Ed., MackPublishing Co., Easton, Pa. (1990)). Preservatives, stabilizers, dyesand even flavoring agents can be provided in the pharmaceuticalcomposition. For example, sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid can be added as preservatives. In addition,antioxidants and suspending agents can be used.

Depending on the target cell, the compounds of the present invention canbe formulated and used as tablets, capsules or elixirs for oral orinhaled administration; salves or ointments for topical application;suppositories for rectal administration; sterile solutions, suspensions,and encapsulated powders and the like for use as inhalants or nasalsprays. Injectables can also be prepared in conventional forms either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions.

Suitable excipients are, for example, water, saline, dextrose, mannitol,lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride andthe like. In addition to those excipients, additional compounds that canbe included in the pharmaceutical compositions described herein eitheralone or in various combinations include Na₂SO₄, MgSO₄, CaCl₂,Histidine, Histine-HCl, and Trehalose or their analogs or Mg saltsand/or Ca salts. These additional compounds can be included in thepharmaceutical compositions to act as excipients or as activeingredients that provide additional beneficial effects. In addition, ifdesired, the injectable pharmaceutical compositions can contain minoramounts of nontoxic auxiliary substances, such as wetting agents, pHbuffering agents and the like.

The pharmaceutically effective amount of a test compound required as adose will depend on the route of administration, the type of animal orpatient being treated, and the physical characteristics of the specificanimal under consideration. The dose can be tailored to achieve adesired effect, such as reduction of elevated levels of mucus in therespiratory tract, but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in themedical arts will recognize. In practicing the methods of the presentinvention, the pharmaceutical compositions can be used alone or incombination with one another, or in combination with other therapeuticor diagnostic agents. These products can be utilized in vivo in anon-human animal subject, in a mammalian subject, in a human subject, orin vitro. In employing them in vivo, the pharmaceutical compositions canbe administered to the patient or subject in a variety of ways,including topically, parenterally, intravenously, subcutaneously,intramuscularly, colonically, rectally, nasally or intraperitoneally,employing a variety of dosage forms. Such methods can also be used intesting the activity of test compounds in vivo.

In some embodiments, these pharmaceutical compositions may be in theform of orally-administrable suspensions, solutions, tablets orlozenges; nasal sprays; inhalants; injectables, topical sprays,ointments, powders, or gels, or formulated into a solutions or drypowders and inhaled with an inhaler.

When administered orally as a suspension, compositions of the presentinvention are prepared according to techniques well-known in the art ofpharmaceutical formulation and may contain microcrystalline cellulosefor imparting bulk, alginic acid or sodium alginate as a suspendingagent, methylcellulose as a viscosity enhancer, and sweeteners/flavoringagents known in the art. As immediate release tablets, thesecompositions may contain microcrystalline cellulose, dicalciumphosphate, starch, magnesium stearate and lactose and/or otherexcipients, binders, extenders, disintegrants, diluents and lubricantsknown in the art. Components in the formulation of a mouthwash or rinseinclude antimicrobials, surfactants, cosurfactants, oils, water andother additives such as sweeteners/flavoring agents known in the art.

When administered by a drinking solution, the composition comprises oneor more of the compounds of the present invention, dissolved in water,with appropriate pH adjustment, and with carrier. The compound can bedissolved in distilled water, tap water, spring water, and the like. ThepH can in some embodiments be adjusted to between about 3.5 and about8.5. Sweeteners can be added, e.g., 1% (w/v) sucrose.

Lozenges can be prepared according to U.S. Pat. No. 3,439,089, hereinincorporated by reference for these purposes.

When administered by nasal aerosol or inhalation, the pharmaceuticalcompositions are prepared according to techniques well-known in the artof pharmaceutical formulation and can be prepared as solutions insaline, employing benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, fluorocarbons, and/orother solubilizing or dispersing agents known in the art. See, forexample, Ansel, H. C. et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, Sixth Ed. (1995). Inhaled powders can also be preparedusing techniques described in U.S. patent application Ser. Nos.11/657,813 and 12/179,520, both of which are incorporated herein byreference in their entirety. These compositions and formulations cangenerally be prepared with suitable nontoxic pharmaceutically acceptableingredients. These ingredients are known to those skilled in thepreparation of nasal dosage forms and some of these can be found inRemington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990, a standard reference in the field. The choice ofsuitable carriers is highly dependent upon the exact nature of the nasaldosage form desired, e.g., solutions, suspensions, ointments, or gels.Nasal dosage forms generally contain large amounts of water in additionto the active ingredient. Minor amounts of other ingredients such as pHadjusters, emulsifiers or dispersing agents, preservatives, surfactants,jelling agents, or buffering and other stabilizing and solubilizingagents can also be present. Generally, the nasal dosage form can beisotonic with nasal secretions.

Nasal formulations can be administers as drops, sprays, aerosols or byany other intranasal dosage form. Optionally, the delivery system can bea unit dose delivery system. The volume of solution or suspensiondelivered per dose can be anywhere from about 5 to about 2000microliters, from about 10 to about 1000 microliters, or from about 50to about 500 microliters. Delivery systems for these various dosageforms can be dropper bottles, plastic squeeze units, atomizers,nebulizers or pharmaceutical aerosols in either unit dose or multipledose packages.

The formulations of this invention can be varied to include; (1) otheracids and bases to adjust the pH; (2) other tonicity imparting agentssuch as sorbitol, glycerin and dextrose; (3) other antimicrobialpreservatives such as other parahydroxy benzoic acid esters, sorbate,benzoate, propionate, chlorbutanol, phenylethyl alcohol, benzalkoniumchloride, and mercurials; (4) other viscosity imparting agents such assodium carboxymethylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, polyvinyl alcohol and other gums; (5) suitableabsorption enhancers; (6) stabilizing agents such as antioxidants, likebisulfite and ascorbate, metal chelating agents such as sodium edetateand drug solubility enhancers such as polyethylene glycols.

One embodiment of the invention includes pharmaceutical compositionsthat at various dosage levels, such as dosage levels between about .01mg and about 100 mg, reduce the quantity of mucus in the respiratorytract of subjects with elevated levels of mucus in their respiratorytracts, and/or that limit increases in the quantity of mucus in therespiratory tract of subjects above a baseline of mucus in theirrespiratory tracts. Examples of such dosage levels include doses ofabout 0.05 mg, 0.06 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 50 mg,or 100 mg. Another embodiment of the invention includes pharmaceuticalcompositions that at various dosage levels, such as dosage levelsbetween about 0.01 mg and about 100 mg, reduce inflammation in therespiratory tract or prevent worsening of inflammation in therespiratory tract. Examples of such dosage levels include doses of about0.05 mg, 0.06 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, or100 mg. The foregoing doses can be administered one or more times perday, for one day, two days, three days, four days, five days, six days,seven days, eight days, nine days, ten days, eleven days, twelve days,thirteen days, or fourteen or more days. Higher doses or lower doses canalso be administered. Typically, dosages can be between about 1 ng/kgand about 10 mg/kg, between about 10 ng/kg and about 1 mg/kg, andbetween about 100 ng/kg and about 100 micrograms/kg. In various examplesdescribed herein, mice were treated with various dosages of thecompositions described herein, including dosages of 0.0008 mg/kg, 0.004mg/kg, 0.02 mg/kg, 0.06 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, and 1.0gm/kg.

In one embodiment a pharmaceutical composition includes DAS181, MgSO₄1.446 mg/ml, CaCl₂ 0.059 mg/ml, Histidine 1.427 mg/ml, Histidine-HCl1.943 mg/ml, and Trehalose 3.000 mg/ml.

In another embodiment a pharmaceutical composition includes DAS181,MgSO₄, CaCl₂, Histidine, Histidine-HCl, and Trehalose.

In another embodiment a pharmaceutical composition includes DAS181,Na₂SO₄, and CaCl₂.

In another embodiment a pharmaceutical composition includes DAS181 andany combination of one or more of the following: Na₂SO₄, MgSO₄, CaCl₂,Histidine, Histidine-HCl, and Trehalose.

In another embodiment a pharmaceutical composition includes (a) anaturally occurring sialidase protein or peptide or an active portionthereof, or a recombinant protein substantially homologous to at least aportion of a naturally occurring sialidase, (b) MgSO₄ 1.446 mg/ml, (c)CaCl₂ 0.059 mg/ml, (d) Histidine 1.427 mg/ml, (e) Histidine-HCl 1.943mg/ml, and (f) Trehalose 3.000 mg/ml. In one embodiment, the protein orpeptide is a sialidase with substantial homology to the A. viscosussialidase (SEQ ID NO:12) or substantial homology to an active portionthereof, such as amino acids 274-666, 274-667, 270-667, 274-681, or290-681 of SEQ ID NO:12, or any other catalytic domain of Actinomycesviscosis sialidase. In other embodiments, the protein or peptide is fromone of the large bacterial sialidases that can degrade the receptorsialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. Forexample, the bacterial sialidase enzymes from Clostridium perfringens(Genbank Accession Number X87369), Arthrobacter ureafaciens, orMicromonospora viridifaciens (Genbank Accession Number D01045) orproteins or peptides that are substantially homologous to thosesialidases or their active portions. In other embodiments, the proteinor peptide is from other sialidases, such as those encoded by the genesNEU2 (SEQ ID NO:8; Genbank Accession Number Y16535; Monti, E, Preti,Rossi, E., Ballabio, A and Borsani G. (1999) Genomics 57:137-143) andNEU4 (SEQ ID NO:9; Genbank Accession Number NM080741; Monti, E, Preti,A, Venerando, B and Borsani, G. (2002) Neurochem Res 27:646-663) (FIG.2), or active portions of those sialidases.

In another embodiment a pharmaceutical composition includes (a) anaturally occurring sialidase protein or peptide or an active portionthereof, or a recombinant protein substantially homologous to at least aportion of a naturally occurring sialidase, (b) MgSO₄, (c) CaCl₂, (d)Histidine, (e) Histidine-HCl, and (f) Trehalose. In one embodiment, theprotein or peptide is a sialidase with substantial homology to the A.viscosus sialidase (SEQ ID NO:12) or substantial homology to an activeportion thereof, such as amino acids 274-666, 274-667, 270-667, 274-681,or 290-681 of SEQ ID NO:12, or any other catalytic domain of Actinomycesviscosis sialidase. In other embodiments, the protein or peptide is fromone of the large bacterial sialidases that can degrade the receptorsialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. Forexample, the bacterial sialidase enzymes from Clostridium perfringens(Genbank Accession Number X87369), Arthrobacter ureafaciens, orMicromonospora viridifaciens (Genbank Accession Number D01045) orproteins or peptides that are substantially homologous to thosesialidases or their active portions. In other embodiments, the proteinor peptide is from other sialidases, such as those encoded by the genesNEU2 (SEQ ID NO:8; Genbank Accession Number Y16535; Monti, E, Preti,Rossi, E., Ballabio, A and Borsani G. (1999) Genomics 57:137-143) andNEU4 (SEQ ID NO:9; Genbank Accession Number NM080741; Monti, E, Preti,A, Venerando, B and Borsani, G. (2002) Neurochem Res 27:646-663) (FIG.2), or active portions of those sialidases.

In another embodiment a pharmaceutical composition includes (a) anaturally occurring sialidase protein or peptide or an active portionthereof, or a recombinant protein substantially homologous to at least aportion of a naturally occurring sialidase, (b) Na₂SO₄, and (c) CaCl₂.In one embodiment, the protein or peptide is a sialidase withsubstantial homology to the A. viscosus sialidase (SEQ ID NO:12) orsubstantial homology to an active portion thereof, such as amino acids274-666, 274-667, 270-667, 274-681, or 290-681 of SEQ ID NO:12, or anyother catalytic domain of Actinomyces viscosis sialidase. In oneembodiment, the protein or peptide is a sialidase with substantialhomology to the A. viscosus sialidase (SEQ ID NO:12) or substantialhomology to an active portion thereof, such as amino acids 274-666,274-667, 270-667, 274-681, or 290-681 of SEQ ID NO:12, or any othercatalytic domain of Actinomyces viscosis sialidase. In otherembodiments, the protein or peptide is from one of the large bacterialsialidases that can degrade the receptor sialic acids Neu5Acalpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For example, the bacterialsialidase enzymes from Clostridium perfringens (Genbank Accession NumberX87369), Arthrobacter ureafaciens, or Micromonospora viridifaciens(Genbank Accession Number D01045) or proteins or peptides that aresubstantially homologous to those sialidases or their active portions.In other embodiments, the protein or peptide is from other sialidases,such as those encoded by the genes NEU2 (SEQ ID NO:8; Genbank AccessionNumber Y16535; Monti, E, Preti, Rossi, E., Ballabio, A and Borsani G.(1999) Genomics 57:137-143) and NEU4 (SEQ ID NO:9; Genbank AccessionNumber NM080741; Monti, E, Preti, A, Venerando, B and Borsani, G. (2002)Neurochem Res 27:646-663) (FIG. 2), or active portions of thosesialidases.

In another embodiment a pharmaceutical composition includes (a) anaturally occurring sialidase protein or peptide or an active portionthereof, or a recombinant protein substantially homologous to at least aportion of a naturally occurring sialidase, and any combination of oneor more of the following: Na₂SO₄, MgSO₄, CaCl₂, Histidine,Histidine-HCl, and Trehalose. In one embodiment, the protein or peptideis a sialidase with substantial homology to the A. viscosus sialidase(SEQ ID NO:12) or substantial homology to an active portion thereof,such as amino acids 274-666, 274-667, 270-667, 274-681, or 290-681 ofSEQ ID NO:12, or any other catalytic domain of Actinomyces viscosissialidase. In other embodiments, the protein or peptide is from one ofthe large bacterial sialidases that can degrade the receptor sialicacids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For example, thebacterial sialidase enzymes from Clostridium perfringens (GenbankAccession Number X87369), Arthrobacter ureafaciens, or Micromonosporaviridifaciens (Genbank Accession Number D01045) or proteins or peptidesthat are substantially homologous to those sialidases or their activeportions. In other embodiments, the protein or peptide is from othersialidases, such as those encoded by the genes NEU2 (SEQ ID NO:8;Genbank Accession Number Y16535; Monti, E, Preti, Rossi, E., Ballabio, Aand Borsani G. (1999) Genomics 57:137-143) and NEU4 (SEQ ID NO:9;Genbank Accession Number NM080741; Monti, E, Preti, A, Venerando, B andBorsani, G. (2002) Neurochem Res 27:646-663) (FIG. 2), or activeportions of those sialidases.

In another embodiment a pharmaceutical composition includes (a) a fusionprotein that has at least one catalytic domain of a sialidase, whereinthe catalytic domain of the sialidase includes the sequence of aminoacids extending from amino acid 274 to amino acid 666 of SEQ ID NO:12(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID NO:12,or any other catalytic domain of Actinomyces viscosis), inclusive, andat least one anchoring domain, wherein the anchoring domain is aglycosaminoglycan (GAG) binding domain of human amphiregulin includingthe amino acid sequence of SEQ ID NO:7, (b) MgSO₄ 1.446 mg/ml, (c) CaCl₂0.059 mg/ml, (d) Histidine 1.427 mg/ml, (e) Histidine-HCl 1.943 mg/ml,and (f) Trehalose 3.000 mg/ml.

In another embodiment a pharmaceutical composition includes (a) a fusionprotein that has at least one catalytic domain of a sialidase, whereinthe catalytic domain of the sialidase includes the sequence of aminoacids extending from amino acid 274 to amino acid 666 of SEQ ID NO:12(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID NO:12,or any other catalytic domain of Actinomyces viscosis), inclusive, andat least one anchoring domain, wherein the anchoring domain is aglycosaminoglycan (GAG) binding domain of human amphiregulin includingthe amino acid sequence of SEQ ID NO:7, (b) MgSO₄, (c) CaCl₂, (d)Histidine, (e) Histidine-HCl, and (f) Trehalose.

In another embodiment a pharmaceutical composition includes (a) a fusionprotein that has at least one catalytic domain of a sialidase, whereinthe catalytic domain of the sialidase includes the sequence of aminoacids extending from amino acid 274 to amino acid 666 of SEQ ID NO:12(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID NO:12,or any other catalytic domain of Actinomyces viscosis), inclusive, andat least one anchoring domain, wherein the anchoring domain is aglycosaminoglycan (GAG) binding domain of human amphiregulin includingthe amino acid sequence of SEQ ID NO:7, (b) Na₂SO₄, and (c) CaCl.

In another embodiment a pharmaceutical composition includes (a) a fusionprotein that has at least one catalytic domain of a sialidase, whereinthe catalytic domain of the sialidase includes the sequence of aminoacids extending from amino acid 274 to amino acid 666 of SEQ ID NO:12(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID NO:12,or any other catalytic domain of Actinomyces viscosis), inclusive, andat least one anchoring domain, wherein the anchoring domain is aglycosaminoglycan (GAG) binding domain of human amphiregulin includingthe amino acid sequence of SEQ ID NO:7, and (b) any combination of oneor more of the following: Na₂SO₄, MgSO₄, CaCl₂, Histidine,Histidine-HCl, and Trehalose.

In another embodiment a pharmaceutical composition includes (a) a fusionprotein having a sialidase or an active portion thereof and an anchoringdomain, (b) MgSO₄ 1.446 mg/ml, (c) CaCl₂ 0.059 mg/ml, (d) Histidine1.427 mg/ml, (e) Histidine-HCl 1.943 mg/ml, and (f) Trehalose 3.000mg/ml.

In another embodiment a pharmaceutical composition includes (a) a fusionprotein having a sialidase or an active portion thereof and an anchoringdomain, (b) MgSO₄, (c) CaCl₂, (d) Histidine, (e) Histidine-HCl, and (f)Trehalose.

In another embodiment a pharmaceutical composition includes (a) a fusionprotein having a sialidase or an active portion thereof and an anchoringdomain, (b) Na₄SO₄, and (c) CaCl₂.

In another embodiment a pharmaceutical composition includes (a) a fusionprotein having a sialidase or an active portion thereof and an anchoringdomain, and (b) any combination of one or more of the following: Na₂SO₄,MgSO₄, CaCl₂,l Histidine, Histidine-HCl, and Trehalose.

Another representative example of a pharmaceutical composition of thepresent invention and that can be used in the methods described hereinincludes the following: DAS181, histidine, magnesium sulfate (or citratesalt), calcium chloride, trehalose, water, Na-Acetate, and acetic acid.

Yet another representative example of a pharmaceutical composition ofthe present invention and that can be used in the methods describedherein includes DAS181 (in any concentration between about 0.01% andabout 100% w/w, between about 1.00% and about 90.0% w/w, between about5.00% and about 80.0% w/w, between about 10.0% and about 70.0% w/w,between about 20.0% and about 70% w/w, between about 30.0% and about70.0% w/w, between about 40.0% and about 70.0% w/w, between about 50.0%and about 70% w/w, between about 60.0% and about 70.0% w/w) incombination with any of the following: histidine or histidine-HCl (inany concentration between about 0.00% and about 90.0% w/w, between about0.01% and about 80.0% w/w, between about 1.00% and about 75.0% w/w,between about 2.00% and about 70.0% w/w, between about 3.00% and about60% w/w, between about 4.00% and about 50.0% w/w, between about 5.00%and about 40.0% w/w, between about 6.00% and about 30% w/w, betweenabout 7.00% and about 20.0% w/w), magnesium sulfate (or citrate salt orsodium sulfate)(in any concentration between about 0.00% and about 90.0%w/w, between about 0.01% and about 80.0% w/w, between about 1.00% andabout 75.0% w/w, between about 2.00% and about 70.0% w/w, between about3.00% and about 60% w/w, between about 4.00% and about 50.0% w/w,between about 5.00% and about 40.0% w/w, between about 6.00% and about30% w/w, between about 7.00% and about 20.0% w/w) , calcium chloride (inany concentration between about 0.00% and about 90.0% w/w, between about0.01% and about 80.0% w/w, between about 0.01% and about 75.0% w/w,between about 0.01% and about 70.0% w/w, between about 0.01% and about60% w/w, between about 0.01% and about 50.0% w/w, between about 0.01%and about 40.0% w/w, between about 0.01% and about 30% w/w, betweenabout 0.10% and about 20.0% w/w), trehalose (in any concentrationbetween about 0.00% and about 90.0% w/w, between about 0.01% and about80.0% w/w, between about 1.00% and about 75.0% w/w, between about 2.00%and about 70.0% w/w, between about 3.00% and about 60% w/w, betweenabout 4.00% and about 50.0% w/w, between about 5.00% and about 40.0%w/w, between about 6.00% and about 30% w/w, between about 7.00% andabout 20.0% w/w), water (in any concentration between about 0.00% andabout 90.0% w/w, between about 0.01% and about 80.0% w/w, between about1.00% and about 75.0% w/w, between about 2.00% and about 70.0% w/w,between about 3.00% and about 60% w/w, between about 4.00% and about50.0% w/w, between about 5.00% and about 40.0% w/w, between about 6.00%and about 30% w/w, between about 7.00% and about 20.0% w/w), Na-Acetate(in any concentration between about 0.00% and about 90.0% w/w, betweenabout 0.01% and about 80.0% w/w, between about 0.01% and about 75.0%w/w, between about 0.01% and about 70.0% w/w, between about 0.01% andabout 60% w/w, between about 0.01% and about 50.0% w/w, between about0.01% and about 40.0% w/w, between about 0.01% and about 30% w/w,between about 0.10% and about 20.0% w/w), and acetic acid (in anyconcentration between about 0.00% and about 90.0% w/w, between about0.01% and about 80.0% w/w, between about 0.01% and about 75.0% w/w,between about 0.01% and about 70.0% w/w, between about 0.01% and about60% w/w, between about 0.01% and about 50.0% w/w, between about 0.01%and about 40.0% w/w, between about 0.01% and about 30% w/w, betweenabout 0.10% and about 20.0% w/w).

Any of the above pharmaceutical compositions may in addition includeMgCl₂ in various concentrations ranging from about 0% to about 75% w/w.

Reducing mucus in the respiratory tract and limiting its increase

Accumulation or elevated levels of mucus in the respiratory airway treecan be caused by an increased volume of mucus produced, and also bydecreased clearance due to defects in the ciliary clearance apparatus inthe respiratory tract. Hypersecretion of mucus can be chronic, butincreased volumes are produced in exacerbations of COPD, during attacksof asthma, and in bronchiectatic and cystic fibrosis patients (W. D.Kim, Eur Respir. J. 1997, 10:1914-1917). Intraluminal mucus accumulation(i.e., elevated levels of mucus) in the airways associated withhypersecretion of mucus or decreased clearance thereof creates aclinical problem in almost all pulmonary diseases and diseases that havean affect on the respiratory tract, including without limitation chronicobstructive pulmonary disease (COPD), bronchitis, bronchiectasis, cysticfibrosis (CF), vasculitis, mucus plugging, Wegener's granulomatosis,pneumonia, tuberculosis, cancer involving the lungs or the respiratorytract, Kartagener syndrome, Young's syndrome, chronic sinopulmonaryinfection, alpha 1-antitrypsin deficiency, primary immunodeficiency,acquired immune deficiency syndrome, opportunistic infection, aninfectious state, a post infectious state, common cold, exercise inducedhypersecretion of mucus, inflammatory bowel disease, ulcerative colitis,Crohn's disease, respiratory infection, respiratory obstruction,inhalation or aspiration of a toxic gas, pulmonary aspiration, oralcoholism. Elevated levels of mucus in the respiratory tract are animportant determinant in the prognosis and clinical features of variouspulmonary diseases, such as chronic bronchitis, bronchiectasis andbronchial asthma, in addition to cystic fibrosis and COPD (W. D. Kim,Eur Respir. J. 1997, 10:1914-1917). Accordingly, in some embodiments,the present disclosure include methods in which a subject with one ormore of these conditions or diseases is selected for treatment. In someembodiments, the methods can include selecting a subject with one ormore of the conditions or diseases provided herein and that is notinfected with one or more of influenza, parainfluenza, and/orrespiratory syncytial virus (RSV). Following selection, the subject canbe treated by administration of one or more of the compositionsdisclosed herein.

Provided herein are methods that include the administration of thecompounds described herein and in U.S. application Ser. Nos. 10/718,986and 10/939,262, or compositions containing them, to reduce the quantityof mucus in the respiratory tract of subjects with elevated levels ofmucus in their respiratory tracts and to limit increases in the quantityof mucus in the respiratory tract of subjects above a baseline of mucusin their respiratory tracts. Thus, the invention relates to method ofusing the therapeutic compounds and/or compositions described herein toprevent or treat diseases that are caused by, cause, or are exacerbatedby respiratory inflammation or increased mucus production, such as, bothallergic and non-allergic asthma, chronic obstructive pulmonary disease(COPD), bronchitis (both acute and non-acute), bronchiectasis, cysticfibrosis (CF), vasculitis, mucuous plugging, Wegener's granulomatosis,and any other disorder that causes inflammation or increased mucusproduction in the respiratory tract or is caused by or exacerbated byinflammation or increased mucus production in the respiratory tract. Theinvention also includes methods of using the therapeutic compoundsand/or compositions described herein to reduce the quantity of mucus inthe respiratory tract of subjects with elevated levels of mucus in theirrespiratory tracts and limit increases in the quantity of mucus in therespiratory tract of subjects above a baseline of mucus in theirrespiratory tracts.

In some embodiments, the methods include administering a composition orcompound containing a therapeutically effective amount of a protein orpeptide having a sialidase or an active portion thereof to a subject.The protein or peptide can be an isolated naturally occurring sialidaseprotein, or a recombinant protein substantially homologous to at least aportion of a naturally occurring sialidase. In one embodiment, apharmaceutical composition or compound contains a sialidase withsubstantial homology to the A. viscosus sialidase (SEQ ID NO:12) orsubstantial homology to an active portion thereof, such as amino acids274-666, 274-667, 270-667, 274-681, or 290-681 of SEQ ID NO:12, or anyother catalytic domain of Actinomyces viscosis sialidase. Thetherapeutically effective amount includes an amount of the protein orpeptide that results in a reduction of the quantity of mucus in therespiratory tract after administration of the composition or compoundwhen compared to the quantity of mucus present prior to administrationof the composition.

In other embodiments, the methods include administering a composition orcompound containing a therapeutically effective amount of a fusionprotein, wherein the fusion protein has at least one catalytic domain ofa sialidase, wherein the catalytic domain of the sialidase includes thesequence of amino acids extending from amino acid 274 to amino acid 666of SEQ ID NO:12 (alternatively, 274 to 666, 270-667, 274-681, 290-681 ofSEQ ID NO:12, or any other catalytic domain of Actinomyces viscosis),inclusive, and at least one anchoring domain, wherein the anchoringdomain is a glycosaminoglycan (GAG) binding domain of human amphiregulinincluding the amino acid sequence of SEQ ID NO:7. The therapeuticallyeffective amount includes an amount of the fusion protein that resultsin a reduction of the quantity of mucus in the respiratory tract afteradministration of the composition or compound when compared to thequantity of mucus present prior to administration of the composition.

In yet other embodiments, the methods include administering acomposition containing a therapeutically effective amount of a fusionprotein having a sialidase or an active portion thereof and an anchoringdomain. The therapeutically effective amount includes an amount of thefusion protein that results in a reduction of the quantity of mucus inthe respiratory tract after administration of the composition orcompound when compared to the quantity of mucus present prior toadministration of the composition.

Other embodiments include methods of preventing, treating orameliorating the effects of chronic obstructive pulmonary disease(COPD), bronchitis, bronchiectasis, cystic fibrosis (CF), vasculitis,mucus plugging, Wegener's granulomatosis, pneumonia, tuberculosis,cancer involving the lungs or the respiratory tract, Kartagenersyndrome, Young's syndrome, chronic sinopulmonary infection, alpha1-antitrypsin deficiency, primary immunodeficiency, acquired immunedeficiency syndrome, opportunistic infection, an infectious state, apost infectious state, common cold, exercise induced hypersecretion ofmucus, inflammatory bowel disease, ulcerative colitis, Crohn's disease,respiratory infection, respiratory obstruction, inhalation or aspirationof a toxic gas, pulmonary aspiration, or alcoholism in a subject with anelevated level of mucus in his or her respiratory tract. The methodsinclude administering (a) a composition containing a therapeuticallyeffective amount of a protein or peptide having a sialidase or an activeportion thereof to a subject, (b) a composition containing atherapeutically effective amount of a fusion protein, wherein the fusionprotein has at least one catalytic domain of a sialidase, wherein thecatalytic domain of the sialidase includes the sequence of amino acidsextending from amino acid 274 to amino acid 666 of SEQ ID NO:12(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID NO:12,or any other catalytic domain of Actinomyces viscosis), inclusive, andat least one anchoring domain, wherein the anchoring domain is aglycosaminoglycan (GAG) binding domain of human amphiregulin includingthe amino acid sequence of SEQ ID NO:7, or (c) a composition or compoundcontaining a therapeutically effective amount of a fusion protein havinga sialidase or an active portion thereof and an anchoring domain. Thetherapeutically effective amount of these compositions or compoundsincludes an amount that results in a reduction of the quantity of mucusin the respiratory tract after administration of the composition whencompared to the quantity of mucus present prior to administration of thecomposition or compound.

Yet other embodiments include methods of limiting an increase in thequantity of mucus in the respiratory tract of a subject above a baselinelevel of mucus in said subject's respiratory tract. The methods includeadministering (a) a composition or compound containing a therapeuticallyeffective amount of a protein or peptide having a sialidase or an activeportion thereof to a subject, (b) a composition or compound containing atherapeutically effective amount of a fusion protein, wherein the fusionprotein has at least one catalytic domain of a sialidase, wherein thecatalytic domain of the sialidase includes the sequence of amino acidsextending from amino acid 274 to amino acid 666 of SEQ ID NO:12(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID NO:12,or any other catalytic domain of Actinomyces viscosis), inclusive, andat least one anchoring domain, wherein the anchoring domain is aglycosaminoglycan (GAG) binding domain of human amphiregulin includingthe amino acid sequence of SEQ ID NO:7, or (c) a composition or compoundcontaining a therapeutically effective amount of a fusion protein havinga sialidase or an active portion thereof and an anchoring domain. Thetherapeutically effective amount of these compositions or compoundsincludes an amount that limits an increase in the quantity of mucus inthe respiratory tract of the subject above a baseline level afteradministration of the composition.

In some embodiments, the compositions or compounds used can includeadditional compounds, including, without limitation, any one or more ofthe following either alone or in various combinations: Na₂SO₄, MgSO₄,CaCl₂, Histidine, Histine-HCl, and Trehalose or their analogs, Mg saltsand/or Ca salts. These additional compounds can be included in thepharmaceutical compositions to act as excipients or as activeingredients that provide additional beneficial effects.

The subjects to be treated with the foregoing methods can be humansubjects or non-human animal subjects. The compounds and compositionsdescribed herein can be administered to epithelial cells of the subjectthrough various routes of administration, including, without limitation,by using inhalers to introduce the compounds or compositions into therespiratory tract of the subject.

In some preferred embodiments, compounds described herein can bedelivered as an inhalant with an inhaler or as a nasal spray. They canalso be administered as eye drops, ear drops, or sprays, ointments,lotions, or gels to be applied to the skin. They can also beadministered intravenously or as a local injection.

Reducing or Preventing Inflammation in the Respiratory Tract

The present invention involves the unexpected discovery thatadministration of the compounds described in U.S. application Ser. Nos.10/718,986 and 10/939,262, or compositions containing them, to reducethe amount of inflammatory cells in the respiratory tract. Thus, theinvention relates to therapeutic compositions or compounds that can beused to reduce inflammation in the respiratory tract or preventworsening of inflammation in the respiratory tract. The invention alsoincludes methods of reducing inflammation in the respiratory tract orpreventing worsening of inflammation in the respiratory tract. Inaddition, the invention relates to therapeutic compositions or compoundsthat can be used to prevent or treat diseases that are caused by, cause,or are exacerbated by respiratory inflammation, such as, both allergicand non-allergic asthma, allergic rhinitis, eczema, psoriasis, reactionsto plant or animal toxins, autoimmune conditions, and any otherdisorder, disease or condition that causes inflammation in therespiratory tract or is caused by or exacerbated by inflammation in therespiratory tract.

In some preferred embodiments, the methods include administering acomposition or compound containing a therapeutically effective amount ofa protein or peptide having a sialidase or an active portion thereof toa subject. The protein or peptide can be an isolated naturally occurringsialidase protein, or a recombinant protein substantially homologous toat least a portion of a naturally occurring sialidase. A preferredpharmaceutical composition contains a sialidase with substantialhomology to the A. viscosus sialidase (SEQ ID NO:12) or substantialhomology to an active portion thereof, such as amino acids 274-666,274-667, 270-667, 274-681, or 290-681 of SEQ ID NO:12, or any othercatalytic domain of Actinomyces viscosis sialidase. The therapeuticallyeffective amount includes an amount of the protein or peptide thatprevents or reduces an allergic or inflammatory response in therespiratory tract the respiratory tract after administration of thecomposition or compound.

In other embodiments, the methods include administering a composition orcompound containing a therapeutically effective amount of a fusionprotein, wherein the fusion protein has at least one catalytic domain ofa sialidase, wherein the catalytic domain of the sialidase includes thesequence of amino acids extending from amino acid 274 to amino acid 666of SEQ ID NO:12 (alternatively, 274 to 666, 270-667, 274-681, 290-681 ofSEQ ID NO:12, or any other catalytic domain of Actinomyces viscosis),inclusive, and at least one anchoring domain, wherein the anchoringdomain is a glycosaminoglycan (GAG) binding domain of human amphiregulinincluding the amino acid sequence of SEQ ID NO:7. The therapeuticallyeffective amount includes an amount of the fusion protein that preventsor reduces an allergic or inflammatory response in the respiratory tractthe respiratory tract after administration of the composition orcompound.

In yet other embodiments, the methods include administering acomposition or compound containing a therapeutically effective amount ofa fusion protein having a sialidase or an active portion thereof and ananchoring domain. The therapeutically effective amount includes anamount of the fusion protein that prevents or reduces an allergic orinflammatory response in the respiratory tract the respiratory tractafter administration of the composition or compound.

In some embodiments, the compositions or compounds used can includeadditional compounds, including, without limitation, any one or more ofthe following either alone or in various combinations: Na₂SO₄, MgSO₄,CaCl₂, Histidine, Histine-HCl, and Trehalose or their analogs. Theseadditional compounds can be included in the pharmaceutical compositionsto act as excipients or as active ingredients that provide additionalbeneficial effects.

The subjects to be treated with the foregoing methods can be humansubjects or non-human animal subjects. The compositions and compoundsdescribed herein can be administered to epithelial cells of the subjectthrough various routes of administration, including, without limitation,by using inhalers to introduce the compounds or compositions into therespiratory tract of the subject.

In some preferred embodiments, compositions or compounds describedherein can be delivered as an inhalant with an inhaler or as a nasalspray. They can also be administered as eye drops, ear drops, or sprays,ointments, lotions, or gels to be applied to the skin. They can also beadministered intravenously or as a local injection.

FIGS. 7A-B show the results of the effect of the use of one of thefusion protein construct depicted in FIG. 5 on inflammatory cells offerrets infected with human unadapted influenza. In ferrets that shedthe virus despite treatment with fusion protein (n=8), the inflammatoryresponse was reduced and animals appeared to be more alert and activecompared to the untreated ferrets that were invariably lethargic andfeverish. For this group of 8 infected, fusion-protein treated animals,the mean AUC (area under the curve) value calculated for the nasalprotein concentrations was reduced by approximately 40% (2.68 vs. 4.48,arbitrary units) compared to the vehicle-treated (phosphate buffersaline) infected animals (FIG. 7B). In vehicle-treated infected animals,the number of inflammatory cells in nasal washes was increased toapproximately 100-fold above those in uninfected animals on day 2 postchallenge. These levels were sustained for 4 additional days. The fusionprotein-treated animals exhibited a significant reduction in the numberof inflammatory cells in the nasal washes. Specifically, the AUC valuefor cell counts was reduced by approximately 3-fold in the fusionprotein-treated animals compared to the vehicle-treated infected animals(1965 vs. 674, arbitrary units, (FIG. 7B). The observed reduction in theinflammatory response indicates the importance of inhibiting viralreplication at the early stage of infection.

Dosage

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and type of patient beingtreated, the particular pharmaceutical composition employed, and thespecific use for which the pharmaceutical composition is employed. Thedetermination of effective dosage levels, that is the dose levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine methods as discussed above. Innon-human animal studies, applications of the pharmaceuticalcompositions are commenced at higher dose levels, with the dosage beingdecreased until the desired effect is no longer achieved or adverse sideeffects are reduced or disappear. The dosage for a compound of thepresent invention can range broadly depending upon the desired affects,the therapeutic indication, route of administration and purity andactivity of the compound. Typically, human clinical applications ofproducts are commenced at lower dosage levels, with dosage level beingincreased until the desired effect is achieved. Alternatively,acceptable in vitro studies can be used to establish useful doses androutes of administration of the test compound. Typically, dosages can bebetween about 1 ng/kg and about 10 mg/kg, between about 10 ng/kg andabout 1 mg/kg, and between about 100 ng/kg and about 100 micrograms/kg.In various examples described herein, mice were treated with variousdosages of the compositions described herein, including dosages of0.0008 mg/kg, 0.004 mg/kg, 0.02 mg/kg, 0.06 mg/kg, 0.1 mg/kg, 0.3 mg/kg,0.6 mg/kg, and 1.0 gm/kg. As nonlimiting examples, the compositionsdescribed herein can be administered to humans in doses of between about0.01 mg and about 100 mg, such as about 0.05 mg, 0.06 mg, 0.1 mg, 0.5mg, 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, or 100 mg, and can be administeredone or more times per day, for one day, two days, three days, four days,five days, six days, seven days, eight days, nine days, ten days, elevendays, twelve days, thirteen days, or fourteen or more days. Higher dosesor lower doses can also be administered. In one embodiment, as shown inExample 3 below, a dose of 0.06 mg/kg of a sialidase compound issufficient to desialylate muscarinic receptors resulting in reducedairway responsiveness to muscarinic receptor agonists, and thuspotentially resulting in reducing airway constriction, airwayhypersensitivity, inflammation, allergies or associated responses, suchas bronchoconstriction, asthma, and mucus overproduction. Efficacy inlow doses, such as 0.06 mg/kg (translating in adult humans into a doseof about 4 or 5 mg), or 0.02 mg/kg (translating in adult humans into adose of about 1 or 2 mg), makes the sialidase-based compounds describedherein good candidates for use in chronic diseases that require repeatedlong-term administration.

A treatment regimen can include administration of the compounds andcompositions described herein from once per day to ten times per day,from once per day to six times per day, from once per day to five timesper day, from once per day to four times per day, from once per day tothree times per day, from once per day to twice per day, and just onceper day. The treatment can last from just one day to daily, weekly,monthly, or other periodic use for a predetermined period of time or forthe remainder of the subject's life.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see,Fingle et al., in The Pharmacological Basis of Therapeutics (1975)). Itshould be noted that the attending physician would know how to and whento terminate, interrupt or adjust administration due to toxicity, organdysfunction or other adverse effects. Conversely, the attendingphysician would also know to adjust treatment to higher levels if theclinical response were not adequate. The magnitude of an administrateddoes in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight and response of the individual patient, including thosefor veterinary applications.

In some preferred regimens, appropriate dosages are administered to eachpatient by either inhaler, nasal spray, or by topical application. Itwill be understood, however, that the specific dose level and frequencyof dosage for any particular patient may be varied and will depend upona variety of factors including the activity of the specific salt orother form employed, the metabolic stability and length of action ofthat compound, the age, body weight, general health, sex, diet, mode andtime of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

In some embodiments, the present disclosure provides methods for usingany one or more of the compositions (indicated below as ‘X’) disclosedherein in the following methods:

Substance X for use as a medicament in the treatment of excess mucus orabnormal (e.g., above normal mucus levels as compared to one or morehealthy subjects (e.g., of the same ethnicity and/or in the same orsimilar geographical location) and/or as indicated by a health carepractitioner), elevated mucus production, and/or any one or more of thediseases/conditions disclosed herein; (each of which is collectivelyreferred to in the following examples as ‘Y.’

Use of substance X for the manufacture of a medicament for the treatmentof Y; and

Substance X for use in the treatment of Y.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1. Effect of Sialidase Treatment on the Early and Late AsthmaticReaction in Guinea Pigs

-   1. Overview

In this study Fludase® was tested in a guinea pig model of allergicasthma. Guinea pigs were sensitised with ovabumin (OVA) or saline andafter 15 or 20 days they were treated with Fludase® or sodium sulfate.On day 21, all the animals were challenged with OVA to measure the earlyasthmatic reaction. Airway compliance/resistance were determined andbroncho-alveolar lavage (BAL) fluid was taken from the left lung tocount the total number of cells and to differentiate them.

-   2. Introduction

The main purpose of this study was to achieve a characterization of theeffect of Fludase® on the early and late reactions in a guinea pig modelfor asthma. The guinea pigs involved in the study were naïve, and thusnot infected with influenza or other infectious agent as part of theexperiment. Asthma was induced by sensitising the guinea pigs on day 0with OVA. After 15 or 20 days the guinea pigs were treated intatrachealwith Fludase® (0.3 mg/kg) or sodium sulfate (0.143 mM, pH 5.0). On day21 the guinea pigs received an OVA aerosol and the airway responsiveness(PenH) was measured. On day 22 the pulmonary resistance and compliancewere determined. At time intervals of 2 minutes doses of histamine from0.2-2 μg/kg were administered by intravenously injection. At the end ofthe experiment guinea pigs were sacrificed and the left lung was lavagedand the isolated BAL cells were washed, counted and differentiated intomacrophages, lymphocytes, neutrophils and eosinophils.

-   3. Materials & Methods-   Animals

Male Hartley-strain guinea pigs (HSD Poc: DH, weighing 400-500) ofspecific pathogen free quality were obtained from Harlan-CPB (Zeist, TheNetherlands). They were used after 1 week of acclimatisation to theirhousing conditions. Water and commercial chow were allowed ad libitum.The experiments were approved by the Animal Ethics Committee of theUtrecht University (Utrecht, The Netherlands).

-   Sensitisation, Pre-treatment & Challenge

Guinea pigs were sensitised with saline (solutions contains 100 mg/mlAl(OH)3) or OVA (solution contains 20 μg/ml OVA and 100 mg/ml Al(OH)3),administered intraperitoneally 0.5 ml and subcutaneously 5 x 0.1 ml,total injection volume 1 ml. After 15 or 20 days animals were treatedonce with 0.3 mg/kg Fludase® and the control animals were treated withsodium sulfate (0.143 mM, pH 5.0) on day 20 by tracheal instillation. Alaryngoscope was used to facilitate the location of the epiglottis. Thenthe Fludase® or sodium sulfate was given with a liquid aerosol using theIA-1C MicroSprayer™ (Penn Century, Inc, Philadelphia, USA). The guineapig was in an upright position during the tracheal instillation.

During this tracheal instillation the guinea pigs were anaesthetizedwith 150 μl of a mixture of Ketamine®, Xylazin®, Atropin and saline(3.5:3:1:3), injected intra muscular in the hind paw.

On day 21 the guinea pigs were challenged by exposure to an aerosol OVA(0.1% wt/vol in sterile saline). The aerosol was generated into a 3liter perspex chamber in which the guinea pigs were placed. First thebasal bronchoconstriction (PenH) was measured. The guinea pigs wereprovoked with OVA aerosol for 10 seconds. Directly after the challengethe early asthmatic reaction was (PenH) was measured.

Allergen-induced early asthmatic reaction in conscious unrestrainedguinea pigs

Airway function of the animals was measured directly after exposure toaerosolised OVA in a ventilated bias flow whole body plethysmograph(Buxco Electronics, Sharon, Conn., USA). The plethysmograph consists ofa reference chamber and an animal chamber. The animal chamber isattached to the outside via a pneumotachograph in the top of theplethysmograph. An aerosol inlet to the animal chamber is centricallylocated in the roof of the animal chamber. When an animal is placed inthe animal chamber and is breathing quietly, it creates pressure betweentidal volume and thoracic movement during respiration. The differentialpressure transducer measures the changes in pressure between animalchamber and the reference chamber and brings these data to apreamplifier. Thereafter, data is sent to a computer where severalparameters are calculated, which represents animal's lung function. Allguinea pigs used were measured basal for 5 minutes and after the aerosolfor 15 minutes in the whole body plethysmograph. Besides known lungfunction parameters as peak expiratory flow (PEF) and tidal volume (TV),the enhanced pause (PenH) was also measured. The formula and explanationof the PenH is shown in FIG. 8. During bronchoconstriction peakexpiratory flow and peak inspiratory flow are increased, whilerelaxation time and expiratory time are decreased. This results in anincreased PenH. Data from bronchoconstriction in conscious unrestrainedguinea pigs are presented in PenH (FIG. 8).

Airway responsiveness in vivo

On day 22 the guinea pigs were anaesthetized with urethane 2 g/kg intraperitoneally. The animals were allowed to breathe spontaneously. Ananaesthesia-induced fall in body temperature was avoided by placing theanimals in a heated chamber, which kept the body temperature at 37° C.The guinea pigs were prepared for the measurement of pulmonaryresistance (R_(L)) and compliance (C) as follows. A small polyethylenecatheter (PE-50) was placed in the right jugular vein for intra venousadministration of increasing doses of histamine (0.2-2 m/kg). First thebasal R_(L) and C were measured for 5 minutes. Thereafter an increasingdose of histamine was injected and R_(L) and C were measured for 2minutes. Airflow and tidal volume were determined by cannulating andconnecting the trachea with Fleisch flow head (nr 000; Meijnhart,Bunnik, The Netherlands) to a pneumotachograph. A pressure transducer(model MP45-2; Validyne Engineering Corp., Northridge, Calif.) measuredthe transpulmonary pressure by determining pressure differences betweenthe tracheal cannula and a cannula filled with saline inserted in theoesophagus. R_(L) and C were determined breath by breath with arespiratory analyser. R_(L) was yielded by dividing transpulmonarypressure by airflow at isovolume points. C was determined by dividingvolume by transpulmonary pressure at isoflow points. Data are presentedas maximal R_(L) and minimal C in cm H₂O/ml*sec⁻¹ an ml/cm H₂O,respectively.

-   Collection of Broncho-alveolar Lung Lavage Cells

Broncho-alveolar lavage cells were obtained as follows. The trachea wastrimmed free of connective tissue and blood vessels and a small incisionwas made for insertion of a cannula into the trachea. The right lung wastied up so only the left lung was lavaged. The left lung was filled with5 ml saline (0.9% NaCl) of 37° C. in situ. Fluid was withdrawn from thelung after gentle massage and collected in a plastic tube on ice (4°C.). This procedure was repeated 3 times (total 15 ml) and the cellsuspensions recovered from each animal were pooled. Thereafter, cellswere sedimented by centrifugation at 1500 rpm for 5 minutes at 4° C. Thesupernatant solution was thrown away and the pellet was resuspended in 1ml saline. Only plastic tubes were used throughout the isolationprocedure in order to minimize adherence of the cells to the walls ofthe tubes.

-   Cell Count and Differentiation

The cells were stained with Turk solution and counted in a BUrker-TUrkbright-line counting chamber (microscope, magnification 100x). Fordifferential BAL cell counts cytospin preparations were made and stainedwith Diff-Quick (Merz & Dade A.G., Dudingen, Switzerland). After codingall cytospin preparations were evaluated by one observer using oilimmersion microscopy (magnification 1000×). Cells were differentiatedinto macrophages, lymphocytes, neutrophils and eosinophils by standardmorphology. At least 200 cells per cytospin preparation were counted andthe absolute number of each cell type was calculated.

-   Statistical Analysis

Unless stated otherwise, data are expressed as arithmeticaverage±standard error of mean and comparisons between groups were madeusing Student's t-test. A probability value p<0.05 was consideredsignificant.

-   4. Results-   Airway Responsiveness in Conscious Unrestrained Guinea Pigs

As shown in FIG. 9, basal airway resistance was not different betweenthe saline guinea pigs treated with sodium sulfate or DAS181 on day 15or day 20 (PenH =0.24±0.009 sal-sodium sulfate, 0.25±0.01 saline-Flu day15, 0.28±0.01 sal-Flu day 20 treated guinea pigs). There was also nodifference between the OVA guinea pigs treated with sodium sulfate orFludase® on day 15 or day 20 at basal level (PenH =0.25±0.01 OVA-sodiumsulfate, 0.27±.02 OVA-Flu day 15, 0.31±0.03 OVA-Flu day 20 treatedguinea pigs).

Ova challenge slightly increased the basal airway resistance in salinesensitized animals (SOD, Flu 15 and 20, FIG. 2). However, the earlyasthmatic reaction in response to the OVA aerosol was strongly increasedin the OVA-sodium sulfate treated guinea pigs (PenH =1.57±0.45). Aftertreatment with Fludase® the early asthmatic reaction was decreased bynearly 30% on day 15, but not on day 20.

-   Cell Count in Broncho-alveolar Lavage Fluid

As shown in FIG. 10, Fludase® decreases the total number of cells bothin saline and OVA guinea pigs.

-   Differential Cell Count in the Broncho-alveolar Lavage Fluid

As shown in FIG. 11, the number of macrophages was enhanced by (30%) inthe OVA guinea pigs compared to the saline treated group. Total numberof macrophages was strongly decreased after treatment with Fludase® onday 15 and 20, both in saline and OVA treated guinea pigs.

As shown in FIG. 12, a similar pattern was observed with the number oflymphocytes. The number of lymphocytes is decreased after Fludase®treatment.

As shown in FIG. 13, compared to historical controls, SOD induces astrong increase in the number of neutrophils into the lungs.Interestingly, Fludase® completely prevented this influx in both thesaline and OVA groups.

As shown in FIG. 14, compared to historical controls SOD induces also aneosinophil influx into the airways, which is further increased by OVAchallenge. Interestingly, Fludase® treatment can restore the number ofeosinophils up to historical control levels.

-   5. Discussion/Conclusion

OVA-sensitised and challenged animals demonstrate an early and lateasthmatic reaction as measured by an increase in PENH up onOVA-challenge, and an increase in the number of inflammatory cells inthe BAL-fluid.

Interestingly, Fludase® (Flu15) reduced the early asthmatic response bynearly 30% (FIG. 9), suggesting an effect of this compound on mast cellstimulation. Moreover, Fludase® had a tremendous effect on theinflammation caused by SOD or OVA. The number of all inflammatory cells(macrophages, lymphocytes, neutrophils and eosinophils) wassignificantly decreased by this compound.

In conclusion, Fludase® demonstrated to be effective in both theSOD-induced inflammation and the OVA-induced inflammation.

Example 2. The Effect of Fludase in a Mouse Model of Acute OVA InducedAsthma

-   1. Introduction

The aim of the study was to investigate whether Fludase® (also referredto as DAS181) (1) inhibits allergen induced airway inflammation andairway hyperreactivity in an acute OVA challenge mouse model of asthma.In addition to studying the effect of DAS 181 alone as an intervention,three other interventions in the mouse asthma model were studiedincluding a) DAS181 +an excipient (used as the dry powder formulation todeliver DAS181 in vivo), b) the excipient alone, and c) dexamethasone(as a comparator).

-   2. Materials and Methods-   Mice

Female BALB/c mice age 12 weeks were purchased from Charles River andhoused in the UCSD vivarium. They were used after approximately one weekof acclimatization in the UCSD vivarium.

OVA sensitization and OVA challenge

Mice were immunized s.c. on days 0, 7, 14, and 21 with 25 ug of OVAadsorbed to 1 mg of alum in 200 ul normal saline to induce a predominantTh2 immune response. Intranasal OVA challenges (20 ug/50 ul) werestarted on day 26 and then repeated on day 28 and day 30. In the no OVAgroup, mice were sensitized to OVA but not challenged intranasally withOVA. Mice had airway responsiveness to methacholine (Mch) measurementsperformed 24 hours after the final OVA challenge by Penh on day 31. Themice involved in the study were naïve and thus not infected withinfluenza or other infectious agent as part of the experiment. Mice werethen immediately sacrificed.

Bronchoalveolar lavage fluid (BAL), blood, and lung tissue wereprocessed for outcomes detailed below.

-   Administration of Test Compounds to Mice-   1. Compounds Tested The following compounds were studied in the    mouse model of asthma-   a) DAS181 with excipient (0.6 mg/kg intranasal): DAS181-F02 (NexBio,    Inc. part # 43-071, lot# 47-034) was prepared in PBS to 20 mgDAS/ml.    Before each dosing, it was freshly diluted in PBS to 0.6 mgDAS181/kg    with dosing volume of 50 uL, for mice with an average body weight of    21 g.-   b) DAS181 (0.6 mg/kg intranasal)-   c) Excipient (50 μL/mouse): Excipient (416TL022A) was supplied by    NexBio, Inc. as a solution, and the concentration of each of the    excipient component is equivalent to that in 20 mgDAS181/ml (MgSO₄    1.446 mg/ml, CaCl₂ 0.059 mg/ml, Histidine 1.427 mg/ml, Histidine-HCl    1.943 mg/ml, and Trehalose 3.000 mg/ml). It was freshly diluted in    PBS for dosing as in a). The final concentration of each excipient    component is equivalent to that in 0.6 mgDAS181/kg, in 50 ul, for    mice with average body weight of 21g.-   d) Dexamethasone (1.0 mg/kg intraperitoneally)

Excipient solution was prepared in the following manner. The targetfinal concentration for each excipient was calculated to reachequivalent concentration when DAS181-F02 bulk dry powder isreconstituted at a 20 mg protein/mL. The 10× stock solutions (100× forCalcium Chloride) for each excipient (10 mL each) were then prepared.Materials used to prepare the stock solutions are listed in Table 1. Allmaterials are USP grade, or equivalent. Appropriate amounts of eachexcipient were weighed into a 15 mL conical tube according to Table 2,and water was added to bring the total weight to 10 grams and vortex todissolve the material completely. The final 1× excipient solution wasprepared by adding 1 mL of each stock solution, and then bringing thevolume to 10 mL using water. All sample preparation was preformedgravimetrically, assuming solution density of 1 gram/mL.

Although referred to as “excipient” or “excipient” solution in theseexamples, these additional compounds can have additional beneficialeffects with respect to reduction of mucus and reduction of inflammationand inflammatory cells. These excipients can also have a synergisticeffect with DAS181.

TABLE 1 Material Information Description Manufacturer Mfg. Part # Mfg.Lot # Expiry L-Histidine Sigma H6034-100 g 078K0179 September 2012L-Histidine Sigma H4036-1 kg 068K8310 January 2012 monohydrochloridemonohydrate a,a-Trehalose, Dihydrate J. T. Baker 4226-04 G47596 No. 2010Magnesium Sulfate EMD 1.05882.0500 K38528682R February 2013 HeptahydrateCalcium Chloride, Dihydrate Mallinckrodt 4616-04 G24475 September 2009WFI B. Braun S9200-SS J8K015 August 2010

TABLE 2 Stock Solution Preparation Sheet (Theoretical) Final conc.Dilution Stock conc. Wt. of Salt for 10 mL Composition (mg/mL) Factor(mg/mL) (mg) MgSO₄ 1.446 10 14.457 296 CaCl₂ 0.059 100 5.943 79Histidine 1.427 10 14.266 143 Histidine•HCl 1.943 10 19.431 213Trehalose 3.000 10 30.000 332

-   2. Mouse Model of Asthma

The following groups of Balb/c mice (n=10 female mice/group) werestudied.

-   a) No OVA-   b) OVA-   c) OVA+DAS181 with excipient-   d) OVA+DAS181-   e) OVA+excipient-   f) OVA+dexamethasone-   3. Timing of Administration of Test Compounds

The test compounds were administered one hour prior to each of the threeintranasal OVA challenges on days 26, 28, and 30.

-   Timing of End-points Studied

Mice were sacrificed 24 hours after the final OVA challenge and blood,BAL, and lungs were analyzed (27).

-   4. End-points Studied-   a) Penh

Airway responsiveness was assessed on day 31, twenty four hours afterthe final OVA inhalation, using a single chamber whole bodyplethysmograph obtained from Buxco (Troy, NY). In this system, anunrestrained, spontaneously breathing mouse is placed into the mainchamber of the plethysmograph, and pressure differences between thischamber and a reference chamber are recorded. The resulting box pressuresignal is caused by volume and resultant pressure changes during therespiratory cycle of the mouse. A low pass filter in the wall of themain chamber allows thermal compensation. From these box pressuresignals, the phases of the respiratory cycle, tidal volumes, and theenhanced pause (Penh) can be calculated. Penh is a dimensionless valuethat represents a function of the proportion of maximal expiratory tomaximal inspiratory box pressure signals and of the timing ofexpiration. It correlates closely with pulmonary resistance measured byconventional two-chamber plethysmography in ventilated mice. In theplethysmograph, mice were exposed for 3 min to nebulized PBS andsubsequently to increasing concentrations of nebulized metacholine(MCh)(3, 6, 12, 24, 48 mg/ml Mch) (Sigma, St. Louis, Mo.) in PBS usingan Aerosonic ultrasonic nebulizer (DeVilbiss). After each nebulization,recordings were taken for 3 min. The Penh values measured during each3-min sequence were averaged and are expressed for each MChconcentration as the percentage of baseline Penh values following PBSexposure.

-   b) Blood Eosinophil Counts

Peripheral blood was collected from mice by cardiac puncture intoEDTA-containing tubes. Erythrocytes were lysed using a 1:10 solution of100 mM potassium carbonate-1.5 M ammonium chloride. The remaining cellswere resuspended in 1 mL PBS. To perform differential cell counts, 200μL resuspended peripheral-blood leukocyte suspensions were cytospun ontomicroscope slides and air-dried. Slides were stained with Wright-Giemsaand the % of eosinophils in the total number of white blood cells wereassessed under a light microscope.

-   c) PAS Staining for Lung Mucus

To quantitate the level of mucus expression in the airway, the number ofperiodic acid Schiff (PAS)-positive and PAS-negative epithelial cells inindividual bronchioles were counted as previously described (Zhang, M.,T. Angata, J. Y. Cho, M. Miller, D. H. Broide, A. Varki. 2007 Blood.109:4280-4287). At least ten bronchioles were counted in each slide.Results are expressed as the percentage of PAS-positive cells perbronchiole, which is calculated from the number of PAS-positiveepithelial cells per bronchus divided by the total number of epithelialcells of each bronchiole. Slides of lung tissue with no OVA, OVA andOVA+DAS181 were also taken and observed.

-   d) MBP Staining of Lungs for Peribronchial Eosinophils

Lungs from the different experimental groups were processed as a batchfor either histologic staining or immunostaining under identicalconditions as described in Zhang et al. Stained and immunostained slideswere all quantified under identical light microscope conditions,including magnification (20×), gain, camera position, and backgroundillumination. Lung sections were processed for MBP immunohistochemistryas described above, using an anti-mouse MBP (Major Basic Protein) Ab(kindly provided by James Lee PhD, Mayo Clinic, Scottsdale, Ariz.) andthe immunoperoxidase method as previously described in Zhang et al.Major Basic Protein is an eosinophil cytoplasmic granule protein whichserves as a marker of eosinophils in tissues. The number of individualcells staining positive for MBP in the peribronchial space were countedusing a light microscope. Results are expressed as the number ofperibronchial cells staining positive for MBP per bronchiole with150-200 μm of internal diameter. At least ten bronchioles were countedin each slide.

-   5. Statistical Analysis

Results in the different groups of mice were compared by Mann Whitneynon-parametric T test. All results are presented as mean+SEM. Astatistical software package (Graph Pad Prism, San Diego, Calif.) wasused for the analysis. P values of <0.05 were considered statisticallysignificant.

6. Results

-   a) Penh

As shown in FIG. 15, OVA challenge induced a significant increase inairway responsiveness as assessed by changes in Penh (OVA vs no OVA;p<0.0001).

OVA challenged mice pre-treated with DAS181+excipient had a significantreduction in Penh compared to OVA challenged mice (OVA vs OVA+DAS181+excipient; p<0.01).

OVA challenged mice pre-treated with DAS181 resulted in a reduction inPenh compared to OVA challenged mice (OVA vs OVA+DAS181; p<0.005).

As shown in FIG. 16, measurement of Penh at 48 mg/ml Mch provides thelargest difference between positive and negative controls (no OVA vsOVA) and is why this dose of Mch is used to assess the effect of anintervention such as DAS181.

-   b) Blood Eosinophils

As shown in FIG. 17, OVA challenge induced a significant increase inblood eosinophils (OVA vs no OVA; p<0.0005).

DAS 181 significantly reduced blood eosinophils (OVA vs OVA+DAS, p=0.04)

-   c) PAS staining for lung mucus

As shown in FIGS. 18-19A-F, OVA challenge induced a significant increasein the % of airway epithelium staining positive for PAS (OVA vs no OVA;p<0.0001).

OVA challenged mice pre-treated with DAS181+excipient had a significantreduction in PAS staining compared to OVA challenged mice (OVA vsOVA+DAS181+ excipient; p<0.0001).

OVA challenged mice pre-treated with DAS181 had a statisticallysignificant reduction in PAS staining compared to OVA challenged mice(OVA vs OVA+DAS181; p<0.0001).

-   Effect on mucus

DAS181 with excipient as well as DAS181 alone significantly reduced PASstaining showing that there is an inhibitory effect of DAS 181 on PASstaining. This shows that DAS181 with excipient or DAS181 alone reducesmucus in the respiratory tract.

-   d) MBP immunostaining of lungs

As shown in FIG. 20, OVA challenge induced a significant increase in thenumber of peribronchial MBP+eosinophils (OVA vs no OVA; p<0.0001).

OVA challenged mice pre-treated with DAS181+excipient had a significantreduction in the number of peribronchial MBP+eosinophils compared to OVAchallenged mice (OVA vs OVA+DAS181+excipient; p=0.02).

Example 3. Reduced Airway Resistance in Naïve Mice Treated Intranasallywith Low Doses of DAS181 (Methacholine Challenged)

The objective of the study was to test the effect of different doselevels of DAS181 on muscarinic receptor mediated airway resistance innaive mice. BALB/c mice (N=4) were treated intranasally with PBS orDAS181 at 0.06, 0.1 or 0.6 mg/kg once daily for three days. Eight hourspost the final treatment animals were challenged with increasing dosesof muscarinic receptor agonist Methacholine (Mch). Airway responsivenesswas assessed using whole body plethysmography. Changes in airwayresistance were expressed as the enhanced pause (Penh), a dimensionlessvalue that represents a function of the proportion of maximal expiratoryto maximal inspiratory box pressure signals and of the timing ofexpiration. Mice were exposed to nebulized PBS and subsequently toincreasing concentrations of nebulized MCh (12, 24, 48 mg/ml Mch) for 2min in PBS. Recordings were performed for 3 min following each exposure.The obtained Penh values were averaged and expressed as the percentageof baseline following PBS exposure.

Results: 24 and 48 mg/ml of the muscarinic receptor agonist methacholineincreased airway resistance above baseline. All animals treated withDAS181 had significantly reduced airway resistance at 48 mg/ml of Mch(Fig). No difference was observed between the different dose groups.

Conclusions: Consistent with previous data, intranasal treatment withDAS181 reduced bronchoconstriction in response to the muscarinicreceptor agonist Mch, further supporting the hypothesis that DAS181dependent desialylation causes a reduction in muscarinic receptorsignaling. Surprisingly, the two higher dose levels did not exert anygreater effect than the lowest dose, suggesting that a dosage level aslow as 0.06 mg/kg of intranasal DAS181 is sufficient to desialylatemuscarinic receptors resulting in reduced airway responsiveness tomuscarinic receptor agonists, and thus potentially resulting in reducinginflammation, allergies or acetylcholine-associated responses, such asbronchoconstriction, asthma, and mucus overproduction. These results aredepicted in FIGS. 21A and 21B.

Example 4. Reduced Airway Resistance in Naïve Mice Treated Intranasallywith a Low Dose of DAS181 (Methacholine Challenged)

The objective of the study was to test the effect of a 0.6 mg/kg oncedaily dose of DAS181 on muscarinic receptor mediated airway resistancein naive mice. BALB/c mice (N=4) were treated intranasally with PBS orDAS181 at 0.6 mg/kg once daily for three days. Eight hours post thefinal treatment animals were challenged with increasing doses ofmuscarinic receptor agonist Methacholine (Mch). Airway responsivenesswas assessed using whole body plethysmography. Changes in airwayresistance were expressed as the enhanced pause (Penh), a dimensionlessvalue that represents a function of the proportion of maximal expiratoryto maximal inspiratory box pressure signals and of the timing ofexpiration. Mice were exposed to nebulized PBS and subsequently toincreasing concentrations of nebulized MCh (3, 6, 12, 24, 48 mg/ml Mch)for 2 min in PBS.

Recordings were performed for 3 min following each exposure. Theobtained Penh values were averaged and expressed as the percentage ofbaseline following PBS exposure.

Results: 3, 6, 12, 24 and 48 mg/ml of the muscarinic receptor agonistmethacholine increased airway resistance above baseline in PBS treatedanimals, and 12, 24 and 48 mg/ml of methacholine increased airwayresistance above baseline in DAS181 treated animals, while 3 and 6 mg/mlof methacholine did not increase airway resistance above baseline inDAS181 treated animals. All animals treated with DAS181 hadsignificantly reduced airway resistance at 6, 12, 24 and 48 mg/ml of Mchcompared to the control.

Conclusions: Consistent with previous data, intranasal treatment withDAS181 reduced bronchoconstriction in response to the muscarinicreceptor agonist Mch, further supporting the hypothesis that DAS181dependent desialylation causes a reduction in muscarinic receptorsignaling. These results are depicted in FIG. 22.

Example 5. Reduced Airway Resistance in Naïve Mice Treated Intranasallywith DAS181 (Carbachol Challenged)

The objective of the study was to test the effect of a 0.6 mg/kg oncedaily dose of DAS181 on muscarinic receptor mediated airway resistancein naive mice. BALB/c mice (N=4) were treated intranasally with PBS orDAS181 at 0.6 mg/kg once daily for three days. Eight hours post thefinal treatment animals were challenged with increasing doses ofmuscarinic receptor agonist carbachol. Airway responsiveness wasassessed using whole body plethysmography. Changes in airway resistancewere expressed as the enhanced pause (Penh), a dimensionless value thatrepresents a function of the proportion of maximal expiratory to maximalinspiratory box pressure signals and of the timing of expiration. Micewere exposed to nebulized PBS and subsequently to increasingconcentrations of nebulized Carbachol (1.25, 2.5, 5, 10, 20 mg/mlcarbachol) for 2 min in PBS. Recordings were performed for 3 minfollowing each exposure. The obtained Penh values were averaged andexpressed as the percentage of baseline following PBS exposure.

Results: 5, 10, and 20 mg/ml of the muscarinic receptor agonistcarbachol increased airway resistance above baseline in both PBS treatedand DAS181 animals. All animals treated with DAS181 had significantlyreduced airway resistance at 5, 10, and 20 mg/ml of carbachol.

Conclusions: Consistent with previous data, intranasal treatment withDAS181 reduced bronchoconstriction in response to the muscarinicreceptor agonist carbachol, further supporting the hypothesis thatDAS181 dependent desialylation causes a reduction in muscarinic receptorsignaling. These results are depicted in FIG. 23.

Example 6. Airway Resistance in Naïve Mice Treated Intranasally with aLow Dose of DAS185 (Methacholine Challenged)

The objective of the study was to test the effect of a 0.6 mg/kg oncedaily dose of DAS185 on muscarinic receptor mediated airway resistancein naive mice. DAS185 is an enzymatically inactive version of DAS181, inwhich a mutation in the sialidase portion renders the sialidaseinactive. BALB/c mice (N=4) were treated intranasally with PBS or DAS185at 0.6 mg/kg once daily for three days. Eight hours post the finaltreatment animals were challenged with increasing doses of muscarinicreceptor agonist Methacholine (Mch). Airway responsiveness was assessedusing whole body plethysmography. Changes in airway resistance wereexpressed as the enhanced pause (Penh), a dimensionless value thatrepresents a function of the proportion of maximal expiratory to maximalinspiratory box pressure signals and of the timing of expiration. Micewere exposed to nebulized PBS and subsequently to increasingconcentrations of nebulized MCh (3, 6, 12, 24, 48 mg/ml Mch) for 2 minin PBS. Recordings were performed for 3 min following each exposure. Theobtained Penh values were averaged and expressed as the percentage ofbaseline following PBS exposure.

Results: There was no difference in airway resistance in response to theMch challenge between the DAS185 treated and PBS treated animals.

Conclusions: Whereas there was a difference in airway resistance betweenDAS181 and PBS treated animals in example 4 above, there was nodifference when DAS181 was replaced with enzymatically inactive DAS185.This experiment shows that reduction in airway resistance in response toDAS181 is sialidase dependent. These results are depicted in FIG. 24.

Example 7. Time-Course of DAS181 Mediated Reduction of Airway Resistance(Methacholine Challenged)

The objective of the study was to test the effect of a 0.6 mg/kg oncedaily dose of DAS181 for one, two or three days on muscarinic receptormediated airway resistance in naive mice. BALB/c mice (N=4) were treatedintranasally with PBS or DAS181 at 0.6 mg/kg once daily for one, two orthree days. Eight hours post the final treatment animals were challengedwith increasing doses of muscarinic receptor agonist Methacholine (Mch).Airway responsiveness was assessed using whole body plethysmography.Changes in airway resistance were expressed as the enhanced pause(Penh), a dimensionless value that represents a function of theproportion of maximal expiratory to maximal inspiratory box pressuresignals and of the timing of expiration. Mice were exposed to nebulizedPBS and subsequently to increasing concentrations of nebulized MCh (3,6, 12, 24, 48 mg/ml Mch) for 2 min in PBS. Recordings were performed for3 min following each exposure. The obtained Penh values were averagedand expressed as the percentage of baseline following PBS exposure.

Results: For one day of treatment, there was no difference in airwayresistance in response to the Mch challenge between the DAS181 treatedand PBS treated animals. For two days of treatment, DAS181 appears toreduce airway resistance relative to PBS at 12 and 24 mg/mlmethacholine, but not at 3, 6 and 48 mg/ml of methacholine. At threedays of treatment, DAS181 had significantly reduced airway resistance at24 and 48 mg/ml of Mch.

Conclusions: Consistent with previous data, there was a difference inairway resistance between DAS181 and PBS treated animals on day 3. Therewas no difference when following one treatment dose, and partialreduction following two days of treatment with DAS181. This experimentshows that 2-3 days of treatment is optimal to achieve a reduction inairway resistance. These results are depicted in FIG. 25.

Example 8. Reduced Airway Resistance in Naïve Mice Treated Intranasallywith Very Low Doses of DAS181 (Methacholine Challenged)

The objective of the study was to test the effect of different low-doselevels of DAS181 on muscarinic receptor mediated airway resistance innaive mice. BALB/c mice (N=4) were treated intranasally with PBS orDAS181 at 0.0008, 0.004, 0.02, or 0.1 mg/kg once daily for three days.Eight hours post the final treatment animals were challenged withincreasing doses of muscarinic receptor agonist Methacholine (Mch).Airway responsiveness was assessed using whole body plethysmography.Changes in airway resistance were expressed as the enhanced pause(Penh), a dimensionless value that represents a function of theproportion of maximal expiratory to maximal inspiratory box pressuresignals and of the timing of expiration. Mice were exposed to nebulizedPBS and subsequently to increasing concentrations of nebulized MCh (12,24, 48 mg/ml Mch) for 2 min in PBS. Recordings were performed for 3 minfollowing each exposure. The obtained Penh values were averaged andexpressed as the percentage of baseline following PBS exposure.

Results: 24 and 48 mg/ml of the muscarinic receptor agonist methacholineincreased airway resistance above baseline. All animals treated withDAS181 had significantly reduced airway resistance at 24 and 48 mg/ml ofMch. No difference was observed between the different dose groups ofDAS181.

Conclusions: Intranasal treatment with very low doses of DAS181 reducedbronchoconstriction in response to the muscarinic receptor agonist Mch,further supporting the hypothesis that DAS181 dependent desialylationcauses a reduction in muscarinic receptor signaling even at very lowdoses of DAS181. This experiment shows that dosage levels as low as0.0008 mg/kg of intranasal DAS181 is sufficient to desialylatemuscarinic receptors resulting in reduced airway responsiveness tomuscarinic receptor agonists, and thus potentially resulting in reducinginflammation, allergies or acetylcholine-associated responses, such asbronchoconstriction, asthma, and mucus overproduction. These results aredepicted in FIG. 26.

Example 9. The Reduced Airway Resistance in Naïve Mice TreatedIntranasally with DAS181 is Dose-Dependent (Methacholine Challenged)

The objective of the study was to test the effect of different doselevels of DAS181 on muscarinic receptor mediated airway resistance innaive mice. BALB/c mice (N=4) were treated intranasally with PBS orDAS181 at 10 ng/kg, 0.1 μg/kg, 1 μg/kg, 10 μg/kg or 0.1 mg/kg once dailyfor three days followed by increasing doses of muscarinic receptoragonist methacholine (Mch). Airway responsiveness was assessed usingwhole body plethysmography. Changes in airway resistance were expressedas the enhanced pause (Penh), a dimensionless value that represents afunction of the proportion of maximal expiratory to maximal inspiratorybox pressure signals and of the timing of expiration. Mice were exposedto nebulized PBS and subsequently to increasing concentrations ofnebulized MCh (12, 24, 48 mg/ml Mch) for 2 min in PBS. Recordings wereperformed for 3 min following each exposure. The obtained Penh valueswere averaged and expressed as the percentage of baseline following PBSexposure.

Results: 24 mg/ml and higher concentrations of the muscarinic receptoragonist methacholine increased airway resistance above baseline. Theairway responsiveness was assessed 8 hours post-treatment on Day 3. Allanimals treated with DAS181 showed reduced airway resistance at 24 mg/mlof Mch, with average percentage change Penh values (representingincrease in airway resistance over baseline) as follows:

Control (PBS): 550-560%

10 ng/kg DAS181: 525-530%

0.1 μg/kg DAS181: 450%

1 μg/kg DAS181: 525%

10 μg/kg DAS181: 225%

0.1 mg/kg DAS181: 200%

As seen from the above results, dose levels of 0.01 or 0.1 mg/kg ofDAS181 significantly reduced airway resistance in response to Mch.

Conclusion: The results demonstrate that reduced airway resistance inresponse to intranasal treatment with DAS181 is dose-dependent.

Example 10. Effect of DAS181 on M2 and M3 Muscarinic Receptor SignalingIn Vitro

The objective of the study was to assess muscarinic receptordesialylation as a potential mechanism for airway protection, using anin vitro model. Human chem-1 cells (Millipore) stably transfected withM2 or M3 muscarinic receptor were treated with 0.4, 2 or 10 μM of DAS181for 30 min at 37° C. prior to the addition of a receptor agonist(acetylcholine; ACh). Receptor signaling was determined using afluorescence reporter for intracellular calcium.

Results: At all doses of DAS181 tested, treatment of human chem-1 cellswith DAS181 increased the potency of agonist-mediated M2 receptorsignaling and decreased the potency of agonist-mediated M3 receptorsignaling. The results are shown below:

A. M2 Receptor Treatment Predicted EC50 Potency Value (M) ACh alone (noDAS181) 390 nM ACh + 10 μM DAS181  140 nM ACh + 2 μM DAS181   130 nMACh + 0.4 μM DAS181 140 nM

B. M3 Receptor Treatment Predicted EC50 Potency Value (M) ACh alone (noDAS181) 6.3 nM ACh + 10 μM DAS181  270 nM ACh + 2 μM DAS181   51 nMACh + 0.4 μM DAS181 110 nM

Conclusion: The results demonstrate that DAS181 increases signalingthrough the M2 muscarinic receptor and decreases signaling through theM3 muscarinic receptor. Thus, the DAS181-mediated responses could beindicative of positive allosteric modulation of the M2, and eitherantagonist or negative modulation M3. Desialylation may offer airwayprotection by reducing the stimulatory signal as well as enhancing theinhibitory signal mediated by muscarinic receptors.

Example 11. Therapeutic Efficacy of DAS181 Microparticle Formulationsagainst Parainfluenza

The efficacy of DAS181 was tested against parainfluenza virus (NV),which is an acute respiratory infection. A 63 year old female patienttested positive for PIV on Jul. 14, 2010 and July 21, 2010; shedding ofthe PIV antigen in nasal swabs and sputum samples collected from thepatient on those days was detected by PCR.

The patient was treated on Jul. 23, 24 and 25, 2010 with one capsule (10mg delivered dose) a day of a dry powder formulation of DAS181 whosecomponents and wt/wt % in the composition are as follows: DAS181:64.54-64.69%; Histidine free base: 4.32-4.60%; Histidine HCl:5.85-6.27%; Trehalose: 9.06-9.68%; Magnesium sulfate: 4.66-5.84%;Calcium chloride: 0.19%; Sodium acetate: 0.04-0.05%; Acetic acid: 0.02%;Water: 10%; Isopropanol: trace amounts. Each capsule contained 13 mg ofthe dry powder in a type 3 clear HPMC capsule (Capsugel), giving adelivered dose of 10 mg. The patient was administered one capsule a dayfor three days (Jul. 23, Jul. 24 and Jul. 25, 2010) by inhalation. Thepatient tested positive for PIV on the day after completion of thetreatment (July 26), and tested negative for PIV on the fifth dayfollowing treatment (July 30, 2010). The results demonstrate theeffectiveness of DAS181 against parainfluenza, i.e., a respiratoryinfection of the upper respiratory tract.

Example 12. Therapeutic Efficacy of DAS181 Microparticle Formulationsagainst Asthma

The efficacy of the DAS181 microparticle formulation used in Example 9above was tested against asthma. A 20 year old male Caucasian asthmapatient was tested for changes in airflow prior to and 1 hour after oraladministration of a 10 mg delivered dose (13 mg capsule) of FormulationA, as measured by FEV1 (forced expiratory volume of air in 1 second).Prior to administration of the drug, the FEV1 of the patient was at 82%of the predicted normal lung function. One hour after administration ofthe drug, the FEV1 of the patient indicated a clinically significantimprovement in lung function, with a 10% increase in value to 92%. Theresults demonstrate that a DAS181 formulation can be effective againstasthma, i.e., a non-infectious respiratory disorder affecting thecentral to upper respiratory tract.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of reducing the quantity or level ofmucus or preventing an increase in the quantity or level of mucus in arespiratory tract of a subject, the method comprising: administering tothe subject a compound or composition comprising a therapeuticallyeffective amount of a fusion protein, wherein the fusion proteincomprises at least one catalytic domain of a sialidase, wherein thecatalytic domain of the sialidase comprises the sequence of amino acidsextending from amino acid 274 to amino acid 666 of SEQ ID NO:12,inclusive, and at least one anchoring domain, wherein the anchoringdomain is a glycosaminoglycan (GAG) binding domain of human amphiregulincomprising the amino acid sequence of SEQ ID NO:7; and thetherapeutically effective amount comprises an amount of the fusionprotein that results in a reduction of the quantity of mucus in therespiratory tract after administration of the compound or compositionwhen compared to the quantity of mucus present prior to administrationof the compound or composition.